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▲ EXPERIMENTAL FIGURE 5-50 In electron microscopy, images are formed from electrons that pass through a specimen or are released from a metal-coated specimen. In a transmission electron microscope (TEM), electrons are extracted from a heated filament, accelerated by an electric field, and focused on the specimen by a magnetic condenser lens. Electrons that pass through the specimen are focused by a series of magnetic objective and projector lenses to form a magnified image of the specimen on a detector, which may be a fluorescent viewing screen, photographic film, or a charged-couple-device (CCD) camera. In a scanning electron microscope (SEM), electrons are focused by condensor and objective lenses on a metal-coated specimen. Scanning coils move the beam across the specimen, and electrons from the metal are collected by a photomultiplier tube detector. In both types of microscopes, because electrons are easily scattered by air molecules, the entire column is maintained at a very high vacuum.

electrons; scattered electrons are not focused by the electromagnetic lenses and do not contribute to the image. Areas that take up less stain appear lighter. Osmium tetroxide preferentially stains certain cellular components, such as membranes (see Figure 5-2a). Specific proteins can be detected in thin sections by the use of electron-dense gold particles coated with protein A, a bacterial protein that binds antibody molecules nonspecifically (Figure 5-51).

Standard electron microscopy cannot be used to study live cells because they are generally too vulnerable to the required conditions and preparatory techniques. In particular, the absence of water causes macromolecules to become de-

natured and nonfunctional. However, the technique of cryo-electron microscopy allows examination of hydrated, unfixed, and unstained biological specimens directly in a transmission electron microscope. In this technique, an aqueous suspension of a sample is applied in an extremely thin film to a grid. After it has been frozen in liquid nitrogen and maintained in this state by means of a special mount, the sample is observed in the electron microscope. The very low temperature (—196 °C) keeps water from evaporating, even in a vacuum, and the sample can be observed in detail in its native, hydrated state without fixing or heavy metal

▲ EXPERIMENTAL FIGURE 5-51 Gold particles coated with protein A are used to detect an antibody-bound protein by transmission electron microscopy. (a) First antibodies are allowed to interact with their specific antigen (e.g., catalase) in a section of fixed tissue. Then the section is treated with a complex of protein A from the bacterium S. aureus and electron-dense gold particles. Binding of this complex to the Fc domains of the antibody molecules makes the location of the target protein, catalase in this case, visible in the electron microscope. (b) A slice of liver tissue was fixed with glutaraldehyde, sectioned, and then treated as described in part (a) to localize catalase. The gold particles (black dots) indicating the presence of catalase are located exclusively in peroxisomes. [From H. J. Geuze et al., 1981, J. Cell Biol. 89:653. Reproduced from the Journal of Cell Biology by copyright permission of The Rockefeller University Press.]

0.5 |m staining. By computer-based averaging of hundreds of images, a three-dimensional model almost to atomic resolution can be generated. For example, this method has been used to generate models of ribosomes (see Figure 4-27), the muscle calcium pump discussed in Chapter 7, and other large proteins that are difficult to crystallize.

Electron Microscopy of Metal-Coated Specimens Can Reveal Surface Features of Cells and Their Components

Transmission electron microscopy is also used to obtain information about the shapes of purified viruses, fibers, enzymes, and other subcellular particles by using a technique, called metal shadowing, in which a thin layer of metal, such

▲ EXPERIMENTAL FIGURE 5-52 Metal shadowing makes surface details on very small particles visible by transmission electron microscopy. The sample is spread on a mica surface and then dried in a vacuum evaporator (1). A filament of a heavy metal, such as platinum or gold, is heated electrically so that the metal evaporates and some of it falls over the sample grid in a very thin film ( 2). To stabilize the replica, the specimen is then coated with a carbon film evaporated from an overhead electrode (3). The biological material is then dissolved by acid ( 4), leaving a metal replica of the sample ( 5), which is viewed in a TEM. In electron micrographs of such preparations, the carbon-coated areas appear light—the reverse of micrographs of simple metal-stained preparations in which the areas of heaviest metal staining appear the darkest.

▲ EXPERIMENTAL FIGURE 5-53 Scanning electron microscopy (SEM) produces a three-dimensional image of the surface of an unsectioned specimen. Shown here is an SEM image of the epithelium lining the lumen of the intestine. Abundant fingerlike microvilli extend from the lumen-facing surface of each cell. The basal lamina beneath the epithelium helps support and anchor it to the underlying connective tissue (Chapter 6). Compare this image of intestinal cells with those in Figure 5-28, a transmission electron micrograph, and in Figure 5-45, a fluorescence micrograph. [From R. Kessel and R. Kardon, 1979, Tissues and Organs, A Text-Atlas of Scanning Electron Microscopy, W. H. Freeman and Company, p. 176.]

as platinum, is evaporated on a fixed and sectioned or rapidly frozen biological sample (Figure 5-52). Acid treatment dissolves away the cell, leaving a metal replica that is viewed in a transmission electron microscope.

Alternatively, the scanning electron microscope allows investigators to view the surfaces of unsectioned metal-coated specimens. An intense electron beam inside the microscope scans rapidly over the sample. Molecules in the coating are excited and release secondary electrons that are focused onto a scintillation detector; the resulting signal is displayed on a cathode-ray tube (see Figure 5-50, right). Because the number of secondary electrons produced by any one point on the sample depends on the angle of the electron beam in relation to the surface, the scanning electron micrograph has a three-dimensional appearance (Figure 5-53). The resolving power of scanning electron microscopes, which is limited by the thickness of the metal coating, is only about 10 nm, much less than that of transmission instruments.

Three-Dimensional Models Can Be Constructed from Microscopy Images

In the past decade, digital cameras have largely replaced optical cameras to record microscopy images. Digital images can be stored in a computer and manipulated by conventional photographic software as well as specialized algorithms. As mentioned earlier, the deconvolution algorithm

can sharpen an image by restoring out-of-focus photons to their origin—an example of a computational method that improves the quality of the image. The details in stored digital images also can be quantified, and objects in images can be reconstructed in three dimensions. For example, the three-dimensional model of an object can be calculated by tomo-graphic methods from a collection of images that cover different views of the object. In light microscopy, a stack of optical sections collected with either a confocal or a decon-volution microscope can be recombined into one three-dimensional image (see Figure 5-49). If a TEM specimen is tilted through various degrees, the resulting images also can be recombined to generate a three-dimensional view of the object (see Figure 5-23).

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