【正文】 microscopes. The following units of length are monly employed in microscopy: 181。 The Plastic ultrathinsections for TEM: 4050nm Sections of LM: 5um。m thick, this is an optical effect。 Range: 15－ 150,000 X. Resolution: 5nm Figure 323. Scanning electron microscopy. Scanning electron micrograph of the stereocilia projecting from a hair cell in the inner ear of a bullfrog (A). For parison, the same structure is shown by differentialinterferencecontrast light microscopy (B) and by thinsection electron microscopy (C). Figure 332. Cells in culture. Scanning electron micrograph of rat fibroblasts growing on the plastic surface of a tissueculture dish. Figure 324. Electron micrographs of individual myosin protein molecules that have been shadowed with platinum. Myosin is a major ponent of the contractile apparatus of muscle. As shown here, it is posed of two globular head regions linked to a mon rodlike tail. III. Metal Shadowing Allows Surface Features to Be Examined Figure 325. Preparation of a metalshadowed replica of the surface of a specimen. Note that the thickness of the metal reflects the surface contours of the original specimen. Figure 326. Freezefracture electron micrograph of the thylakoid membranes from the chloroplast of a plant cell. These membranes, which carry out photosynthesis, are stacked up in multiple layers. The largest particles seen in the membrane are the plete photosystem IIa plex of multiple proteins. IV. FreezeFracture and FreezeEtch Electron Microscopy Figure 327. Freezeetch electron microscopy. The specimen is rapidly frozen, and the block of ice is fractured with a knife (A). The ice level is then lowered by sublimation in a vacuum, exposing structures in the cell that were near the fracture plane (B). Following these steps, a replica of the still frozen surface is prepared, and this is examined in a transmission electron microscope. ?Freeze –Fracture Replication and Freeze Etching quick freeze deep etching Figure 328. Regular array of protein filaments in an insect muscle. To obtain this image, the muscle cells were rapidly frozen to liquid helium temperature, fractured through the cytoplasm, and subjected to deep etching. A metalshadowed replica was then prepared and examined at high magnification. (Courtesy of Roger Cooke and John Heuser.) Quickfreeze, deepetch electron microscopy of processes in MAP2 (a), MAP2C (b) or tau (c) transfected Sf9 cells, and microtubules copolymerized in vitro with either MAP2 (d) or tau (e). Figure 329. Electron micrograph of negatively stained actin filaments. Each filament is about 8 nm in diameter and is seen, on close inspection, to be posed of a helical chain of globular actin molecules. (Courtesy of Roger Craig.) V. Negative Staining and Cryoelectron Microscopy Allow Macromolecules to Be Viewed at High Resolution Figure 1031. The threedimensional structure of a bacteriorhodopsin molecule. The polypeptide chain crosses the lipid bilayer as seven a helices. The location of the chromophore and the probable pathway taken by protons during the lightactivated pumping cycle are shown. When activated by a photon, the chromophore is thought to pass an H+ to the side chain of aspartic acid 85 (pink sphere marked 85). Subsequently, three other H+ transfers are thought to plete the cyclefrom aspartic acid 85 to the extracellular space, from aspartic acid 96 (pink sphere marked 96) to the chromophore, and from the cytosol to aspartic acid 96. (R. Henderson et al. J. Mol. :899929) 3. Isolating Cells and Growing Them in Culture Figure 331. A fluorescenceactivated cell sorter. When a cell passes through the laser beam, it is monitored for fluorescence. Droplets containing single cells are given a negative or positive charge, depending on whether the cell is fluorescent or not. The droplets are then deflected by an electric field into collection tubes according to their charge. Note that the cell concentration must be adjusted so that most droplets contain no cells and flow to a waste container together with any cell clumps. The same apparatus can also be used to separate fluorescently labeled chromosomes from one another, providing valuable starting material for the isolation and mapping of genes. Figure 332. Cells in culture. Scanning electron micrograph of rat fibroblasts growing on the plastic surface of a tissueculture dish. (Courtesy of Guenter AlbrechtBuehler.) Figure 333. The production of hybrid cells. Human cells and mouse cells are fused to produce heterocaryons, which eventually form hybrid cells. These particular hybrid cells are useful for mapping human genes on specific human chromosomes because most of the human chromosomes are quickly lost in a random manner, leaving clones that retain only one or a few. The hybrid cells produced by fusing other types of cells often retain most of their chromosomes. 4. The Fractionation and analysis for cell’s contents A. The technique of differential centrifugation Stepbystep procedure for the pu