Figure 3-23. Scanning electron microscopy. Scanning electron micrograph of the stereocilia projecting from a hair cell in the inner ear of a bullfrog (A). For comparison, the same structure is shown by differential-interference-contrast light microscopy (B) and by thin-section electron microscopy (C). Figure 3-32. Cells in culture. Scanning electron micrograph of rat fibroblasts growing on the plastic surface of a tissue-culture dish. Figure 3-24. Electron micrographs of individual myosin protein molecules that have been shadowed with platinum. Myosin is a major component of the contractile apparatus of muscle. As shown here, it is composed of two globular head regions linked to a common rodlike tail. III. Metal Shadowing Allows Surface Features to Be Examined Figure 3-25. Preparation of a metal-shadowed replica of the surface of a specimen. Note that the thickness of the metal reflects the surface contours of the original specimen. Figure 3-26. Freeze-fracture 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 complete photosystem II-a complex of multiple proteins. IV. Freeze-Fracture and Freeze-Etch Electron Microscopy Figure 3-27. Freeze-etch 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 3-28. 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 metal-shadowed replica was then prepared and examined at high magnification. (Courtesy of Roger Cooke and John Heuser.) Quick-freeze, deep-etch 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 3-29. Electron micrograph of negatively stained actin filaments. Each filament is about 8 nm in diameter and is seen, on close inspection, to be composed 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 10-31. The three-dimensional 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 light-activated 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 complete the cyclefrom aspartic acid 85 to the extra-cellular 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. Biol.213:899-929) 3. Isolating Cells and Growing Them in Culture Figure 3-31. A fluorescence-activated 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 3-32. Cells in culture. Scanning electron micrograph of rat fibroblasts growing on the plastic surface of a tissue-culture dish. (Courtesy of Guenter Albrecht-Buehler.) Figure 3-33. 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 Step-by-step procedure for the purification of organelles by differential centrifugation. S=(dx/dt)/ 2x =1 10-13sec.  Figure 3-34. The preparative ultracentrifuge. Figure 3-35. Cell fractionation by centrifugation. Repeated centrifugation at progressively higher speeds will fractionate homogenates of cells into their components. In general, the smaller the subcellular component, the greater is the centrifugal force required to sediment it. Typical values for the various centrifugation steps referred to in the figure arelow speed: 1,000 times gravity for 10 minutes medium speed: 20,000 times gravity for 20 minutes high speed: 80,000 times gravity for 1 hour very high speed: 150,000 times gravity for 3 hours Figure 3-36. Comparison of methods of velocity sedimentation and equilibrium sedimentation. Figure 3-37. The separation of small molecules by paper chromatography. After the sample has been applied to one end of the paper (the "origin") and dried, a solution containing a mixture of two or more solvents is allowed to flow slowly through the paper by capillary action. Different components in the sample move at different rates in the paper according to their relative solubility in the solvent that is preferentially adsorbed onto the fibers of the paper. B. Paper chromatography Figure 3-38. The separation of molecules by column chromatography. The sample is applied to the top of a cylindrical glass or plastic column filled with a permeable solid matrix, such as cellulose, immersed in solvent. Then a large amount of solvent is pumped slowly through the column and is collected in separate tubes as it emerges from the bottom. Various components of the sample travel at different rates through the column and are thereby fractionated into different tubes. C. Column chromatography Figure 3-39. Three types of matrices used for chromatography. In ion-exchange chromatography (A) the insoluble matrix carries ionic charges that retard molecules of opposite charge. Matrices commonly used for separating proteins are DEAE-cellulose, which is positively charged, and CM-cellulose and phosphocellulose, which are negatively charged. In gel-filtration chromatography (B) the matrix is inert but porous. Molecules that are small enough to penetrate into the matrix are thereby delayed and travel more slowly through the column. Beads of cross-linked polysaccharide (dextran or agarose) are available commercially in a wide range of pore sizes, making them suitable for the fractionation of molecules of various molecular weights, from less than 500 to more than 5 x 106. Affinity chromatography (C) utilizes an insoluble matrix that is covalently linked to a specific ligand, such as an antibody molecule or an enzyme substrate, that will bind a specific protein. Figure 3-40. Protein purification by chromatography. In this example a homogenate of cells was first fractionated by allowing it to percolate through an ion-exchange resin packed into a column (A). The column was washed, and the bound proteins were then eluted by passing a solution containing a gradually increasing concentration of salt onto the top of the column. Proteins with the lowest affinity for the ion-exchange resin passed directly through the column and were collected in the earliest fractions eluted from the bottom of the column. The remaining proteins were eluted in sequence according to