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 autoradiographs, in which the developed silver grains appear white in a dark background. Clinically, dark-field microscopy is useful in examining urine for crystals, such as those of uric acid and oxalate, and in demonstrating specific bacteria such as spirochetes, particularly Treponema pallidum, the microorganism 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, the microscope’s most widespread application is the display of introduced fluores cence, as in the detection of antigens or antibodies in immunocytochemical staining procedures (see Fig. 1.6). 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 band 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 allows visualization of a biologic specimen in three dimensions. The two lenses in the confocal microscope (objective and phototube lens) are perfectly aligned to focus light from the focal point of one lens to the focal point of the other lens. The major difference between a conventional and a confocal microscope is the addition of a detector aperture (pinhole) that is conjugate with the focal point of the lens; therefore, it is confocal. This precisely positioned pinhole allows only “in-focus” light to pass into a photomultiplier (detector) device, whereas the “out-of-focus” light is blocked from entering the detector (Fig. 1.11). This system
FIGURE 1.11 • Diagram of the in-focus and out-of-focus emitted light in the confocal microscope. a. This diagram shows the path of the laser beam and emitted light when the imaging structure is directly at the focus of the lens. The screen with a pinhole at the other side of the optical system of the confocal microscope allows the light from the structure in focus to pass through the pinhole. The light is then translated into an image by computer software. Because the focal point of the objective lens of the microscope forms a sharp image at the level at which the pinhole is located, these two points are referred to as confocal points. b. This diagram shows the path of the laser beam and the emitted light, which is out of focus in relation to the pinhole. Thus, the light from the specimen that gets blocked by the pinhole is never detected.