Light Microscope

scanning beam electron detector projection lens lens specimen objective lens ocular lens image in eye



limage on viewing screen

In dark-field microscopy, no direct light from the light source is gathered by the objective lens

In dark-field microscopy, only light that has been scattered or diffracted by structures in the specimen reaches the objective. The dark-field microscope is equipped with a special condenser that illuminates the specimen with strong, oblique light. Thus, the field of view appears as a dark background on which small particles in the specimen that reflect some light into the objective appear bright.

The effect is similar to that of dust particles seen in the light beam emanating from a slide projector in a darkened room. The light reflected off the dust particles reaches the retina of the eye, thus making the particles visible.

The resolution of the dark-field microscope cannot be better than that of the bright-field microscope, using, as it does, the same wavelength source. Smaller individual particles can be detected in dark-field images, however, because of the enhanced contrast that is created.

The dark-field microscope is useful in examining au-toradiographs, in which the developed silver grains appear white in a dark background. Clinically, it is useful in examining urine for crystals, such as those of uric acid and oxalate, and in demonstrating spirochetes, particularly Treponema pallidum, the organism that causes syphilis, a sexually transmitted disease.

The fluorescence microscope makes use of the ability of certain molecules to fluoresce under ultraviolet light

A molecule that fluoresces emits light of wavelengths in the visible range when exposed to an ultraviolet (UV) source. The fluorescence microscope is used to display naturally occurring fluorescent (autofluorescent) molecules, such as vitamin A and some neurotransmitters. Because autofluorescent molecules are not numerous, however, its most widespread application is the display of introduced fluorescence, as in the detection of antigens or antibodies in immunocytochemical staining procedures (see Fig. 1.4). Specific fluorescent molecules can also be injected into an animal or directly into cells and used as tracers. Such methods have been useful in studying intercellular (gap) junctions, in tracing the pathway of nerve fibers in neurobiology, and in detecting fluorescent growth markers of mineralized tissues.

Various filters are inserted between the UV light source and the specimen to produce monochromatic or near-monochromatic (single-wavelength or narrow-band-wavelength) light. A second set of filters inserted between the specimen and the objective allows only the narrow ba nd of wavelength of the fluorescence to reach the eye or to reach a photographic emulsion or other analytic processor.

The confocal scanning microscope combines components of a light optical microscope with a scanning system to dissect a specimen optically

The confocal scanning microscope is a relatively new microscope system used to study the structure of biologic materials. The illuminating laser light system that it uses is strongly convergent and therefore produces a shallow scanning spot. The light emerging from the spot is directed to a photomultiplier tube, where it is analyzed. A mirror system is used to move the laser beam across the specimen, illuminating a single spot at a time (Fig. 1.8). The data from each point of the specimen scanned by this moving spot are recorded and stored in a computer. This information is then displayed on a high-resolution video monitor to create a visual image. The major advantage of this system is its ability to visualize a specimen in very thin optical sections (approximately 1 /xm thick). The out-of-focus regions are subtracted from the image by the computer program, thus creating an extremely sharp image. In these aspects, confocal microscopy resembles the imaging process in computed axial tomography (x-ray) scanning (CAT scans). Ordinary or nonconfocal light imaging contains superimposed in-focus and out-of-focus specimen parts, thereby reducing image quality.

Furthermore, by using only the narrow depth of the in-focus image, it is possible to create multiple images at varying depths within the specimen. Thus, one can literally dissect layer by layer through the thickness of the specimen. It is also possible to use the computer to make three-dimensional reconstructions of a series of these images. Because each individual image located at a specific depth within the specimen is extremely sharp, the resulting assembled three-dimensional image is equally sharp.

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