1. The Light Microscope
The resolving power of the light microscope under ideal conditions is about half the wavelength of the light being used. (Resolving power is the distance that must separate two point sources of light if they are to be seen as two distinct images.) With yellow light of a wavelength of 0.4 ![]()
m, the smallest separable diameters are thus about 0.2 ![]()
m, ie, one-third the width of a typical prokaryotic cell. The useful magnification of a microscope is the magnification that makes visible the smallest resolvable particles. Several types of light microscopes are commonly used in microbiology:
2. Bright-Field Microscope
The bright-field microscope is most commonly used in microbiology courses and consists of two series of lenses (objective and ocular lens), which function together to resolve the image. These microscopes generally employ a 100-power objective lens with a 10-power ocular lens, thus magnifying the specimen 1000 times. Particles 0.2 ![]()
m in diameter are therefore magnified to about 0.2 mm and so become clearly visible. Further magnification would give no greater resolution of detail and would reduce the visible area (field).
With this microscope, specimens are rendered visible because of the differences in contrast between them and the surrounding medium. Many bacteria are difficult to see well because of their lack of contrast with the surrounding medium. Dyes (stains) can be used to stain cells or their organelles and increase their contrast so that they can be more easily seen in the bright-field microscope.
3. Phase Contrast Microscope
The phase contrast microscope was developed to improve contrast differences between cells and the surrounding medium, making it possible to see living cells without staining them; with bright-field microscopes, killed and stained preparations must be used. The phase contrast microscope takes advantage of the fact that light waves passing through transparent objects, such as cells, emerge in different phases depending on the properties of the materials through which they pass. This effect is amplified by a special ring in the objective lens of a phase contrast microscope, leading to the formation of a dark image on a light background.
4. Dark-Field Microscope
The dark-field microscope is a light microscope in which the lighting system has been modified to reach the specimen from the sides only. This is accomplished through the use of a special condenser that both blocks direct light rays and deflects light off a mirror on the side of the condenser at an oblique angle. This creates a "dark field" that contrasts against the highlighted edge of the specimens and results when the oblique rays are reflected from the edge of the specimen upward into the objective of the microscope. Resolution by dark-field microscopy is quite high. Thus, this technique has been particularly useful for observing organisms such as Treponema pallidum, a spirochete which is less than 0.2 ![]()
m in diameter and therefore cannot be observed with a bright-field or phase contrast microscope.
5. Fluorescence Microscope
The fluorescence microscope is used to visualize specimens that fluoresce, which is the ability to absorb short wavelengths of light (ultraviolet) and give off light at a longer wavelength (visible). Some organisms fluoresce naturally because of the presence within the cells of naturally fluorescent substances such as chlorophyll. Those that do not naturally fluoresce may be stained with a group of fluorescent dyes called fluorochromes. Fluorescense microscopy is widely used in clinical diagnostic microbiology. For example, the fluorochrome auramine O, which glows yellow when exposed to ultraviolet light, is strongly absorbed by Mycobacterium tuberculosis, the bacterium that causes tuberculosis. When the dye is applied to a specimen suspected of containing M tuberculosis and exposed to ultraviolet light, the bacterium can be detected by the appearance of bright yellow organisms against a dark background.
The principal use of fluorescence microscopy is a diagnostic technique called the fluorescent-antibody (FA) technique or immunofluorescence. In this technique, specific antibodies (eg, antibodies to Legionella pneumophila) are chemically labeled with a fluorochrome such as fluorescein isothiocyanate (FITC). These fluorescent antibodies are then added to a microscope slide containing a clinical specimen. If the specimen contains L pneumophila, the fluorescent antibodies will bind to antigens on the surface of the bacterium, causing it to fluoresce when exposed to ultraviolet light.
Differential Interference Contrast (DIC) Microscope
Differential interference contrast microscopes employ a polarizer to produce polarized light. The polarized light beam passes through a prism that generates two distinct beams; these beams pass through the specimen and enter the objective lens where they are recombined into a single beam. Because of slight differences in refractive index of the substances each beam passed through, the combined beams are not totally in phase but instead create an interference effect, which intensifies subtle differences in cell structure. Structures such as spores, vacuoles, and granules appear three dimensional. DIC microscopy is particularly useful for observing unstained cells because of its ability to generate images that reveal internal cell structures that are less apparent by bright-field techniques.
6.The Electron Microscope
The high resolving power of the electron microscope has enabled scientists to observe the detailed structures of prokaryotic and eukaryotic cells. The superior resolution of the electron microscope is due to the fact that electrons have a much shorter wavelength than the photons of white light.
There are two types of electron microscopes in general use: the transmission electron microscope (TEM), which has many features in common with the light microscope, and the scanning electron microscope (SEM). The TEM was the first to be developed and employs a beam of electrons projected from an electron gun and directed or focused by an electromagnetic condenser lens onto a thin specimen. As the electrons strike the specimen, they are differentially scattered by the number and mass of atoms in the specimen; some electrons pass through the specimen and are gathered and focused by an electromagnetic objective lens, which presents an image of the specimen to the projector lens system for further enlargement. The image is visualized by allowing it to impinge on a screen that fluoresces when struck with the electrons. The image can be recorded on photographic film. TEM can resolve particles 0.001 ![]()
m apart. Viruses, with diameters of 0.01–0.2 ![]()
m, can be easily resolved.
The SEM generally has a lower resolving power than the TEM; however, it is particularly useful for providing three-dimensional images of the surface of microscopic objects. Electrons are focused by means of lenses into a very fine point. The interaction of electrons with the specimen results in the release of different forms of radiation (eg, secondary electrons) from the surface of the material, which can be captured by an appropriate detector, amplified, and then imaged on a television screen.
An important technique in electron microscopy is the use of "shadowing." This involves depositing a thin layer of heavy metal (such as platinum) on the specimen by placing it in the path of a beam of metal ions in a vacuum. The beam is directed at a low angle to the specimen, so that it acquires a "shadow" in the form of an uncoated area on the other side. When an electron beam is then passed through the coated preparation in the electron microscope and a positive print is made from the "negative" image, a three-dimensional effect is achieved (eg, see Figure 2–22).Confocal Scanning Laser Microscope
The confocal scanning laser microscope (CSLM) couples a laser light source to a light microscope. In confocal scanning laser microscopy, a laser beam is bounced off a mirror that directs the beam through a scanning device. Then the laser beam is directed through a pinhole that precisely adjusts the plane of focus of the beam to a given vertical layer within the specimen. By precisely illuminating only a single plane of the specimen, illumination intensity drops off rapidly above and below the plane of focus, and stray light from other planes of focus are minimized. Thus, in a relatively thick specimen, various layers can be observed by adjusting the plane of focus of the laser beam.
Cells are often stained with fluorescent dyes to make them more visible. Alternatively, false color images can be generated by adjusting the microscope in such a way as to make different layers take on different colors. The CSLM is equipped with computer software to assemble digital images for subsequent image processing. Thus, images obtained from different layers can be stored and then digitally overlaid to reconstruct a three-dimensional image of the entire specimen.(Jawetz, Melnick, & Adelberg's Medical Microbiology, 24th Edition by Vishal )
