H. Lodish - Molecular Cell Biology (5ed, Freeman, 2003) (796244), страница 79
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In a scanning electron microscope(SEM), electrons are focused by condensor and objective lenseson a metal-coated specimen. Scanning coils move the beamacross the specimen, and electrons from the metal are collectedby a photomultiplier tube detector. In both types of microscopes,because electrons are easily scattered by air molecules, theentire 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.
Areasthat 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 inthin sections by the use of electron-dense gold particlescoated with protein A, a bacterial protein that binds antibody molecules nonspecifically (Figure 5-51).Standard electron microscopy cannot be used to studylive 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-0.5 µm▲ EXPERIMENTAL FIGURE 5-51 Gold particles coatedwith protein A are used to detect an antibody-bound proteinby transmission electron microscopy.
(a) First antibodies areallowed to interact with their specific antigen (e.g., catalase) in asection of fixed tissue. Then the section is treated with acomplex of protein A from the bacterium S. aureus and electrondense gold particles. Binding of this complex to the Fc domainsof the antibody molecules makes the location of the targetprotein, 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 localizecatalase. The gold particles (black dots) indicating the presence ofcatalase are located exclusively in peroxisomes.
[From H. J. Geuzeet al., 1981, J. Cell Biol. 89:653. Reproduced from the Journal of CellBiology by copyright permission of The Rockefeller University Press.]192CHAPTER 5 • Biomembranes and Cell Architecturestaining. By computer-based averaging of hundreds of images, a three-dimensional model almost to atomic resolutioncan be generated. For example, this method has been usedto generate models of ribosomes (see Figure 4-27), the muscle calcium pump discussed in Chapter 7, and other largeproteins that are difficult to crystallize.AbsorptiveepithelialcellsElectron Microscopy of Metal-CoatedSpecimens Can Reveal Surface Featuresof Cells and Their ComponentsBasallaminaTransmission 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, suchSampleMica surface1Evaporated platinumMetal replicaMicrovilli5 m▲ EXPERIMENTAL FIGURE 5-53 Scanning electronmicroscopy (SEM) produces a three-dimensional image ofthe surface of an unsectioned specimen.
Shown here is anSEM image of the epithelium lining the lumen of the intestine.Abundant fingerlike microvilli extend from the lumen-facingsurface of each cell. The basal lamina beneath the epitheliumhelps support and anchor it to the underlying connective tissue(Chapter 6). Compare this image of intestinal cells with those inFigure 5-28, a transmission electron micrograph, and in Figure5-45, 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.]2Evaporated carbonCarbon film3Acid4Metal replica readyfor visualization5▲ EXPERIMENTAL FIGURE 5-52 Metal shadowing makessurface details on very small particles visible by transmissionelectron microscopy. The sample is spread on a mica surfaceand then dried in a vacuum evaporator ( 1 ). A filament of a heavymetal, such as platinum or gold, is heated electrically so that themetal evaporates and some of it falls over the sample grid in avery thin film ( 2 ). To stabilize the replica, the specimen is thencoated with a carbon film evaporated from an overhead electrode( 3 ). The biological material is then dissolved by acid ( 4 ), leavinga metal replica of the sample ( 5 ), which is viewed in a TEM.
Inelectron micrographs of such preparations, the carbon-coatedareas appear light—the reverse of micrographs of simple metalstained preparations in which the areas of heaviest metalstaining appear the darkest.as platinum, is evaporated on a fixed and sectioned or rapidly frozen biological sample (Figure 5-52). Acid treatmentdissolves away the cell, leaving a metal replica that is viewedin a transmission electron microscope.Alternatively, the scanning electron microscope allows investigators to view the surfaces of unsectioned metal-coatedspecimens. An intense electron beam inside the microscopescans rapidly over the sample. Molecules in the coating areexcited and release secondary electrons that are focused ontoa scintillation detector; the resulting signal is displayed on acathode-ray tube (see Figure 5-50, right).
Because the number of secondary electrons produced by any one point on thesample depends on the angle of the electron beam in relation to the surface, the scanning electron micrograph has athree-dimensional appearance (Figure 5-53). The resolvingpower of scanning electron microscopes, which is limited bythe thickness of the metal coating, is only about 10 nm,much less than that of transmission instruments.Three-Dimensional Models Can Be Constructedfrom Microscopy ImagesIn the past decade, digital cameras have largely replaced optical cameras to record microscopy images.
Digital imagescan be stored in a computer and manipulated by conventional photographic software as well as specialized algorithms. As mentioned earlier, the deconvolution algorithmKey Termscan sharpen an image by restoring out-of-focus photons totheir origin—an example of a computational method thatimproves the quality of the image. The details in stored digital images also can be quantified, and objects in images canbe reconstructed in three dimensions.
For example, the threedimensional model of an object can be calculated by tomographic methods from a collection of images that coverdifferent views of the object. In light microscopy, a stack ofoptical sections collected with either a confocal or a deconvolution microscope can be recombined into one threedimensional image (see Figure 5-49). If a TEM specimen istilted through various degrees, the resulting images also canbe recombined to generate a three-dimensional view of theobject (see Figure 5-23).KEY CONCEPTS OF SECTION 5.6Visualizing Cell ArchitectureThe limit of resolution of a light microscope is about200 nm; of a scanning electron microscope, about 10 nm;and of a transmission electron microscope, about 0.1 nm.■Because cells and tissues are almost transparent, varioustypes of stains and optical techniques are used to generatesufficient contrast for imaging.■Phase-contrast and differential interference contrast(DIC) microscopy are used to view the details of live, unstained cells and to monitor cell movement.■In immunofluorescence microscopy, specific proteinsand organelles in fixed cells are stained with fluorescencelabeled monoclonal antibodies.
Multiple proteins can belocalized in the same sample by staining with antibodieslabeled with different fluorochromes.■When proteins tagged with naturally occurring green fluorescent protein (GFP) or its variants are expressed in livecells, they can be visualized in a fluorescence microscope.■With the use of dyes whose fluorescence is proportionalto the concentration of Ca2 or H ions, fluorescence microscopy can measure the local concentration of Ca2 ionsand intracellular pH in living cells.■Confocal microscopy and deconvolution microscopy usedifferent methods to optically section a specimen, therebyreducing the blurring due to out-of-focus fluorescencelight.
Both methods provide much sharper images, particularly of thick specimens, than does standard fluorescencemicroscopy.PERSPECTIVES FOR THE FUTUREAdvances in bioengineering will make major contributionsnot only to our understanding of cell and tissue function butalso to the quality of human health.
In a glass slide consistingof microfabricated wells and channels, for example, reagentscan be introduced and exposed to selected parts of individualcells; the responses of the cells can then be detected by lightmicroscopy and analyzed by powerful image-processing software. These types of studies will lead to discovery of newdrugs, detection of subtle phenotypes of mutant cells (e.g.,tumor cells), and development of comprehensive models ofcellular processes. Bioengineers also are fabricating artificialtissues based on a synthetic three-dimensional architectureincorporating layers of different cells.
Eventually such artificial tissues will provide replacements for defective tissues insick, injured, or aging individuals.Microscopy will continue to be a major tool in cell biology, providing images that relate to both the chemistry(i.e.,interactions among proteins) and the mechanics (i.e., movements) involved in various cell processes. The forces causingmolecular and cellular movements will be directly detectedby fluorescent sensors in cells and the extracellular matrix.Improvements to high-resolution imaging methods will permit studies of single molecules in live cells, something thatis currently possible only in vitro. Finally, cells will be studied in more natural contexts, not on glass coverslips but in3D gels of extracellular matrix molecules.
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