《环境微生物学与实验》课程教学资源（文献资料）The Study of Microbial Structure - Microscopy and Specimen Preparation.pdf_大学文库

Prescott−Harley−Klein: Microbiology, Fifth Edition I. Introduction to Microbiology 2. The Study of Microbial Structure: Microscopy and Specimen Preparation © The McGraw−Hill Companies, 2002 Concepts 1. Light microscopes use glass lenses to bend and focus light rays and produce enlarged images of small objects. The resolution of a light microscope is determined by the numerical aperture of its lens system and by the wavelength of the light it employs; maximum resolution is about 0.2 m. 2. The most common types of light microscopes are the bright-field, darkfield, phase-contrast, and fluorescence microscopes. Each yields a distinctive image and may be used to observe different aspects of microbial morphology. 3. Because most microorganisms are colorless and therefore not easily seen in the bright-field microscope, they are usually fixed and stained before observation. Either simple or differential staining can be used to enhance contrast. Specific bacterial structures such as capsules, endospores, and flagella also can be selectively stained. 4. The transmission electron microscope achieves great resolution (about 0.5 nm) by using electron beams of very short wavelength rather than visible light. Although one can prepare microorganisms for observation in other ways, one normally views thin sections of plastic-embedded specimens treated with heavy metals to improve contrast. 5. External features can be observed in great detail with the scanning electron microscope, which generates an image by scanning a fine electron beam over the surface of specimens rather than projecting electrons through them. 6. New forms of microscopy are improving our ability to observe microorganisms and molecules. Two examples are the confocal scanning laser microscope and the scanning probe microscope. There are more animals living in the scum on the teeth in a man’s mouth than there are men in a whole kingdom. —Antony van Leeuwenhoek Microbiology usually is concerned with organisms so small they cannot be seen distinctly with the unaided eye. Because of the nature of this discipline, the microscope is of crucial importance. Thus it is important to understand how the microscope works and the way in which specimens are prepared for examination. The chapter begins with a detailed treatment of the standard bright-field microscope and then describes other common types of light microscopes. Next preparation and staining of specimens for examination with the light microscope are discussed. This is followed by a description of transmission and scanning electron microscopes, both of which are used extensively in current microbiological research. The chapter closes with a brief introduction to two newer forms of microscopy: scanning probe microscopy and confocal microscopy. 2.1 Lenses and the Bending of Light To understand how a light microscope operates, one must know something about the way in which lenses bend and focus light to form images. When a ray of light passes from one medium to another, refraction occurs—that is, the ray is bent at the interface. The refractive index is a measure of how greatly a substance 18 Chapter 2 The Study of Microbial Structure: Microscopy and Specimen Preparation slows the velocity of light, and the direction and magnitude of bending is determined by the refractive indexes of the two media forming the interface. When light passes from air into glass, a medium with a greater refractive index, it is slowed and bent toward the normal, a line perpendicular to the surface (figure 2.1). As light leaves glass and returns to air, a medium with a lower refractive index, it accelerates and is bent away from the normal. Thus a prism bends light because glass has a different refractive index from air, and the light strikes its surface at an angle. Lenses act like a collection of prisms operating as a unit. When the light source is distant so that parallel rays of light strike the lens, a convex lens will focus these rays at a specific point, the focal point (F in figure 2.2). The distance between the center of the lens and the focal point is called the focal length (f in figure 2.2). Our eyes cannot focus on objects nearer than about 25 cm or 10 inches (table 2.1). This limitation may be overcome by using a convex lens as a simple magnifier (or microscope) and holding it close to an object. A magnifying glass provides a clear image at much closer range, and the object appears larger. Lens strength is related to focal length; a lens with a short focal length will magnify an object more than a weaker lens having a longer focal length. 4 3 2 1 θ θ θ θ Figure 2.1 The Bending of Light by a Prism. Normals (lines perpendicular to the surface of the prism) are indicated by dashed lines. As light enters the glass, it is bent toward the first normal (angle 2 is less than 1). When light leaves the glass and returns to air, it is bent away from the second normal (4 is greater than 3). As a result the prism bends light passing through it. f F Figure 2.2 Lens Function. A lens functions somewhat like a collection of prisms. Light rays from a distant source are focused at the focal point F. The focal point lies a distance f, the focal length, from the lens center.

Prescott−Harley−Klein: Microbiology, Fifth Edition I. Introduction to Microbiology 2. The Study of Microbial Structure: Microscopy and Specimen Preparation © The McGraw−Hill Companies, 2002 1. Define refraction, refractive index, focal point, and focal length. 2. Describe the path of a light ray through a prism or lens. 3. How is lens strength related to focal length? 2.2 The Light Microscope Microbiologists currently employ a variety of light microscopes in their work; bright-field, dark-field, phase-contrast, and fluorescence microscopes are most commonly used. Modern microscopes are all compound microscopes. That is, the magnified image formed by the objective lens is further enlarged by one or more additional lenses. The Bright-Field Microscope The ordinary microscope is called a bright-field microscope because it forms a dark image against a brighter background. The microscope consists of a sturdy metal body or stand composed of a base and an arm to which the remaining parts are attached (figure 2.3). A light source, either a mirror or an electric illuminator, is located in the base. Two focusing knobs, the fine and coarse adjustment knobs, are located on the arm and can move either the stage or the nosepiece to focus the image. The stage is positioned about halfway up the arm and holds microscope slides by either simple slide clips or a mechanical stage clip. A mechanical stage allows the operator to move a slide around smoothly during viewing by use of stage control knobs. The substage condenser is mounted within or beneath the stage and focuses a cone of light on the slide. Its position often is fixed in simpler microscopes but can be adjusted vertically in more advanced models. The curved upper part of the arm holds the body assembly, to which a nosepiece and one or more eyepieces or oculars are attached. More advanced microscopes have eyepieces for both eyes and are called binocular microscopes. The body assembly itself contains a series of mirrors and prisms so that the barrel holding the eyepiece may be tilted for ease in viewing (figure 2.4). The nosepiece holds three to five objectives with lenses of differing magnifying power and can be rotated to position any objective beneath the body assembly. Ideally a microscope should 2.2 The Light Microscope 19 Table 2.1 Common Units of Measurement Unit Abbreviation Value 1 centimeter cm 102 meter or 0.394 inches 1 millimeter mm 103 meter 1 micrometer m 106 meter 1 nanometer nm 109 meter 1 Angstrom Å 1010 meter Ocular (eyepiece) Body Arm Coarse focus adjustment knob Fine focus adjustment knob Stage adjustment knobs Interpupillary adjustment Nosepiece Objective lens (4) Mechanical stage Substage condenser Aperture diaphragm control Base with light source Field diaphragm lever Light intensity control Figure 2.3 A Bright-Field Microscope. The parts of a modern bright-field microscope. The microscope pictured is somewhat more sophisticated than those found in many student laboratories. For example, it is binocular (has two eyepieces) and has a mechanical stage, an adjustable substage condenser, and a built-in illuminator.

Prescott−Harley−Klein: Microbiology, Fifth Edition I. Introduction to Microbiology 2. The Study of Microbial Structure: Microscopy and Specimen Preparation © The McGraw−Hill Companies, 2002 be parfocal—that is, the image should remain in focus when objectives are changed. The path of light through a bright-field microscope is shown in figure 2.4. The objective lens forms an enlarged real image within the microscope, and the eyepiece lens further magnifies this primary image. When one looks into a microscope, the enlarged specimen image, called the virtual image, appears to lie just beyond the stage about 25 cm away. The total magnification is calculated by multiplying the objective and eyepiece magnifications together. For example, if a 45 objective is used with a 10 eyepiece, the overall magnification of the specimen will be 450. Microscope Resolution The most important part of the microscope is the objective, which must produce a clear image, not just a magnified one. Thus resolution is extremely important. Resolution is the ability of a lens to separate or distinguish between small objects that are close together. Much of the optical theory underlying microscope design was developed by the German physicist Ernst Abbé in the 1870s. The minimum distance (d) between two objects that reveals them as separate entities is given by the Abbé equation, in which lambda () is the wavelength of light used to illuminate the specimen and n sin is the numerical aperture (NA). 0.5 d _____ n sin As d becomes smaller, the resolution increases, and finer detail can be discerned in a specimen. The preceding equation indicates that a major factor in resolution is the wavelength of light used. The wavelength must be shorter than the distance between two objects or they will not be seen clearly. Thus the greatest resolution is obtained with light of the shortest wavelength, light at the blue end of the visible spectrum (in the range of 450 to 500 nm). The electromagnetic spectrum of radiation (p. 130). The numerical aperture (n sin ) is more difficult to understand. Theta is defined as 1 2 the angle of the cone of light entering an objective (figure 2.5). Light that strikes the microorganism after passing through a condenser is cone-shaped. When this cone has a narrow angle and tapers to a sharp point, it does not spread out much after leaving the slide and therefore does not adequately separate images of closely packed objects. The resolution is low. If the cone of light has a very wide angle and spreads out rapidly after passing through a specimen, closely packed objects appear widely separated and are resolved. The angle of the cone of light that can enter a lens depends on the refractive index (n) of the medium in which the lens works, as well as upon the objective itself. The refractive index for air is 1.00. Since sin cannot be greater than 1 (the maximum is 90° and sin 90° is 1.00), no lens working in air can have a numerical aperture greater than 1.00. The only practical way to raise the numerical aperture above 1.00, and therefore achieve higher resolution, is to increase the refractive index with immersion oil, a colorless liquid with the same refractive index as glass (table 2.2). If air is replaced with immersion oil, many light rays that did not enter the objective due to reflection and refraction at the surfaces of the objective lens and slide will now do so (figure 2.6). An increase in numerical aperture and resolution results. 20 Chapter 2 The Study of Microbial Structure: Microscopy and Specimen Preparation Light path Figure 2.4 A Microscope’s Light Path. The light path in an advanced bright-field microscope and the location of the virtual image are shown. (See also figure 2.23.) Objective Working distance Slide with specimen θ θ Figure 2.5 Numerical Aperture in Microscopy. The angular aperture is 1 2 the angle of the cone of light that enters a lens from a specimen, and the numerical aperture is n sin . In the right-hand illustration the lens has larger angular and numerical apertures; its resolution is greater and its working distance smaller.

Prescott−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 resolution of a microscope depends upon the numerical aperture of its condenser as well as that of the objective. This is evident from the equation describing the resolution of the complete microscope. dmicroscope ______________________ (NAobjective NAcondenser) Most microscopes have a condenser with a numerical aperture between 1.2 and 1.4. However, the condenser numerical aperture will not be much above about 0.9 unless the top of the condenser is oiled to the bottom of the slide. During routine microscope operation, the condenser usually is not oiled and this limits the overall resolution, even with an oil immersion objective. The limits set on the resolution of a light microscope can be calculated using the Abbé equation. The maximum theoretical resolving power of a microscope with an oil immersion objective (numerical aperture of 1.25) and blue-green light is approximately 0.2 m. (0.5)(530 nm) d –––––––––––– 212 nm or 0.2 m 1.25 At best, a bright-field microscope can distinguish between two dots around 0.2 m apart (the same size as a very small bacterium). Normally a microscope is equipped with three or four objectives ranging in magnifying power from 4 to 100 (table 2.2). The working distance of an objective is the distance between the front surface of the lens and the surface of the cover glass (if one is used) or the specimen when it is in sharp focus. Objectives with large numerical apertures and great resolving power have short working distances. The largest useful magnification increases the size of the smallest resolvable object enough to be visible. Our eye can just detect a speck 0.2 mm in diameter, and consequently the useful limit of magnification is about 1,000 times the numerical aperture of the objective lens. Most standard microscopes come with 10 eyepieces and have an upper limit of about 1,000 with oil immersion. A 15 eyepiece may be used with good objectives to achieve a useful magnification of 1,500. Any further magnification increase does not enable a person to see more detail. A light microscope can be built to yield a final magnification of 10,000, but it would simply be magnifying a blur. Only the electron microscope provides sufficient resolution to make higher magnifications useful. Proper specimen illumination also is extremely important in determining resolution. A microscope equipped with a concave mirror between the light source and the specimen illuminates the slide with a fairly narrow cone of light and has a small numerical aperture. Resolution can be improved with a substage condenser, a large light-gathering lens used to project a wide cone of light through the slide and into the objective lens, thus increasing the numerical aperture. The Dark-Field Microscope Living, unstained cells and organisms can be observed by simply changing the way in which they are illuminated. A hollow cone of light is focused on the specimen in such a way that unreflected and unrefracted rays do not enter the objective. Only light that has been reflected or refracted by the specimen forms an image (figure 2.7). The field surrounding a specimen appears black, while the object itself is brightly illuminated 2.2 The Light Microscope 21 Table 2.2 The Properties of Microscope Objectives Objective Property Scanning Low Power High Power Oil Immersion Magnification 4× 10× 40–45× 90–100× Numerical aperture 0.10 0.25 0.55–0.65 1.25–1.4 Approximate focal length (f) 40 mm 16 mm 4 mm 1.8–2.0 mm Working distance 17–20 mm 4–8mm 0.5–0.7 mm 0.1 mm Approximate resolving power with light 2.3 µm 0.9 µm 0.35 µm 0.18 µm of 450 nm (blue light) Air Oil Cover glass Slide Figure 2.6 The Oil Immersion Objective. An oil immersion objective operating in air and with immersion oil

Prescott−Harley−Klein: Microbiology, Fifth Edition I. Introduction to Microbiology 2. The Study of Microbial Structure: Microscopy and Specimen Preparation © The McGraw−Hill Companies, 2002 (figure 2.8a,b); because the background is dark, this type of microscopy is called dark-field microscopy. Considerable internal structure is often visible in larger eucaryotic microorganisms (figure 2.8b). The dark-field microscope is used to identify bacteria like the thin and distinctively shaped Treponema pallidum (figure 2.8a), the causative agent of syphilis. The Phase-Contrast Microscope Unpigmented living cells are not clearly visible in the brightfield microscope because there is little difference in contrast between the cells and water. Thus microorganisms often must be fixed and stained before observation to increase contrast and create variations in color between cell structures. A phase-contrast microscope converts slight differences in refractive index and cell density into easily detected variations in light intensity and is an excellent way to observe living cells (figure 2.8c–e). The condenser of a phase-contrast microscope has an annular stop, an opaque disk with a thin transparent ring, which produces a hollow cone of light (figure 2.9). As this cone passes through a cell, some light rays are bent due to variations in density and refractive index within the specimen and are retarded by about 1 4 wavelength. The deviated light is focused to form an image of the object. Undeviated light rays strike a phase ring in the phase plate, a special optical disk located in the objective, while the deviated rays miss the ring and pass through the rest of the plate. If the phase ring is constructed in such a way that the undeviated light passing through it is advanced by 1 4 wavelength, the deviated and undeviated waves will be about 1 2 wavelength out of 22 Chapter 2 The Study of Microbial Structure: Microscopy and Specimen Preparation Dark-field stop Abbé condenser Specimen Objective Figure 2.7 Dark-Field Microscopy. The simplest way to convert a microscope to dark-field microscopy is to place (a) a dark-field stop underneath (b) the condenser lens system. The condenser then produces a hollow cone of light so that the only light entering the objective comes from the specimen. (a) (b)

Prescott−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 similar 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 specimen, while the reference beam passes through a clear area of the slide. After passing through the specimen, the two beams are combined and interfere with each other to form an image. A live, unstained 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 because 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 excited molecule as it gives up its trapped energy and returns to a more stable state. The fluorescence microscope (figure 2.12) exposes a specimen 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 darkfield 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 darkphase-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)

Prescott−Harley−Klein: Microbiology, Fifth Edition I. Introduction to Microbiology 2. The Study of Microbial Structure: Microscopy and Specimen Preparation © The McGraw−Hill Companies, 2002 from the light emitted when they fluoresce (figure 2.13). A barrier filter positioned after the objective lenses removes any remaining ultraviolet light, which could damage the viewer’s eyes, or blue and violet light, which would reduce the image’s contrast. The fluorescence microscope has become an essential tool in medical microbiology and microbial ecology. Bacterial pathogens (e.g., Mycobacterium tuberculosis, the cause of tuberculosis) can be identified after staining them with fluorochromes or specifically labeling them with fluorescent antibodies using immunofluorescence procedures. In ecological studies the fluorescence microscope is used to observe microorganisms stained with fluorochrome-labeled probes or fluorochromes such as acridine orange and DAPI (diamidino-2- phenylindole, a DNA-specific stain). The stained organisms will fluoresce orange or green and can be detected even in the midst of other particulate material. It is even possible to distinguish live bacteria from dead bacteria by the color they fluoresce after treatment with a special mixture of stains (figure 2.13d). Thus the microorganisms can be viewed and directly counted in a relatively undisturbed ecological niche. Immunofluorescence and diagnostic microbiology (pp. 781, 831–32). 26 Chapter 2 The Study of Microbial Structure: Microscopy and Specimen Preparation 6. Barrier filter Removes any remaining exciter wavelengths (up to about 500 nm) without absorbing longer wavelengths of fluorescing objects 5. Specimen stained with fluorochrome Emits fluorescence when activated by exciting wavelength of light 4. Dark-field condenser Provides dark background for fluorescence Mirror 3. Exciter filter Allows only short wavelength light (about 400 nm ) through 2. Heat filter 1. Mercury vapor arc lamp Eyepiece Objective lens Figure 2.12 Fluorescence Microscopy. The principles of operation of a fluorescence microscope