hao” nen the two bear vacuolesand cucaryotic ucare caryi The Fluorescence microscope The micre ores thus far considered nroduce an i ave a lon Figure 2.11 Differential Interference Contrast Micr The fluorescence micros cope(figure 2.12)exposes a spec lamp or other source produces an intens The Differential Interference Contrast Microscope The)microscope is sim oen whic of brightly upon exposure to light of te luonchmme-labcledPrescott−Harley−Klein: Microbiology, Fifth Edition I. Introduction to Microbiology 2. The Study of Microbial Structure: Microscopy and Specimen Preparation © The McGraw−Hill Companies, 2002 The Differential Interference Contrast Microscope The differential interference contrast (DIC) microscope is simi￾lar to the phase-contrast microscope in that it creates an image by detecting differences in refractive indices and thickness. Two beams of plane polarized light at right angles to each other are generated by prisms. In one design, the object beam passes through the speci￾men, while the reference beam passes through a clear area of the slide. After passing through the specimen, the two beams are com￾bined and interfere with each other to form an image. A live, un￾stained specimen appears brightly colored and three-dimensional (figure 2.11). Structures such as cell walls, endospores, granules, vacuoles, and eucaryotic nuclei are clearly visible. The Fluorescence Microscope The microscopes thus far considered produce an image from light that passes through a specimen. An object also can be seen be￾cause it actually emits light, and this is the basis of fluorescence microscopy. When some molecules absorb radiant energy, they become excited and later release much of their trapped energy as light. Any light emitted by an excited molecule will have a longer wavelength (or be of lower energy) than the radiation originally absorbed. Fluorescent light is emitted very quickly by the ex￾cited molecule as it gives up its trapped energy and returns to a more stable state. The fluorescence microscope (figure 2.12) exposes a spec￾imen to ultraviolet, violet, or blue light and forms an image of the object with the resulting fluorescent light. A mercury vapor arc lamp or other source produces an intense beam, and heat transfer is limited by a special infrared filter. The light passes through an exciter filter that transmits only the desired wavelength. A dark￾field condenser provides a black background against which the fluorescent objects glow. Usually the specimens have been stained with dye molecules, called fluorochromes, that fluoresce brightly upon exposure to light of a specific wavelength, but some microorganisms are autofluorescing. The microscope forms an image of the fluorochrome-labeled microorganisms 2.2 The Light Microscope 25 Phase plate Bacterium Ray deviated by specimen is 1/4 wavelength out of phase. Deviated ray is 1/2 wavelength out of phase. Deviated and undeviated rays cancel each other out. Figure 2.10 The Production of Contrast in Phase Microscopy. The behavior of deviated and undeviated or undiffracted light rays in the dark￾phase-contrast microscope. Because the light rays tend to cancel each other out, the image of the specimen will be dark against a brighter background. Figure 2.11 Differential Interference Contrast Microscopy. A micrograph of the protozoan Amoeba proteus. The three-dimensional image contains considerable detail and is artificially colored (160)