【正文】 tides are made. The cells must therefore use a bypass pathway to synthesize their nucleic acids, and this pathway is defective in the mutant cell line to which the normal B lymphocytes are fused. Because neither cell type used for the initial fusion can grow on its own, only the hybrid cells survive. 7. Monoclonal Antibodies 8. Gene Knockout mice Mario Capecchi (Late 1980s) (University of Utah) embryonic stem cells in inner cell mass as target cells 1/104 cells undergo a process of homologous rebination. 9. The technique for the take apart and gather up of cell, and microscope manipulation ?Preparation and reform of karyoplast and cytoplast ?Transgenic animals and plants Transgenic mice 10 weeks 44g and 29g THANKS! 。 (197。 B, courtesy of J. Hajdu and I. Andersson。 ?Greatly increase the contrast of an image so that very small objects bee visible. Figure 313. The confocal microscope. This diagram shows that the basic arrangement of optical ponents is similar to that of the standard fluorescence microscope except that a laser is used to illuminate a small pinhole whose image is focused at a single point in the specimen (A). Fluorescence from this focal point in the specimen is focused at a second pinhole (B). Light from elsewhere in the specimen is not focused here and therefore does not contribute to the final image (C). By scanning the beam of light across the specimen, a very sharp twodimensional image of the exact plane of focus is built up that is not significantly degraded by light from other regions of the specimen. F. The confocal microscope GFP can be used to study dynamic processes as they occur in a living cell. Figure 314. Comparison of conventional and confocal fluorescence microscopy. These two micrographs are of the same intact gastrulastage Drosophila embryo that has been stained with a fluorescent probe for actin filaments. The conventional, unprocessed image (A) is blurred by the presence of fluorescent structures above and below the plane of focus. In the confocal image (B), this outoffocus information is removed, which results in a crisp optical section of the cell in the embryo. Figure 316. Limit of resolution of the electron microscope. Electron micrograph of a thin layer of gold showing the individual files of atoms in the crystal as bright spots. The distance between adjacent files of gold atoms is about nm (2 197。 Criticalpoint drying。 Sections of TEM: 100nm Figure 319. Diagram of the copper grid used to support the thin sections of a specimen in the transmission electron microscope. Thin sections Figure 320. Electron micrograph of a roottip cell stained with osmium and other heavy metal ions. The cell wall, nucleus, vacuoles, mitochondria, endoplasmic reticulum, Golgi apparatus, and ribosomes are easily seen. Figure 321. Electron micrograph of a cell showing the location of a particular enzyme (nucleotide diphosphatase) in the Golgi apparatus. A thin section of the cell was incubated with a substrate that formed an electrondense precipitate upon reaction with the enzyme Figure 363. Immunogold electron microscopy. Electron micrographs of an insulinsecreting cell in which the insulin molecules have been labeled with antiinsulin antibodies bound to tiny colloidal gold spheres. Most of the insulin is stored in the dense cores of secretory vesicles。 The Plastic ultrathinsections for TEM: 4050nm Sections of LM: 5um。 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