【正文】 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。 (197。 Criticalpoint drying。 they are, in reality, only onetenth this diameter. (Courtesy of P. Sammeh and G. Borisy.) Figure 359. Methods to introduce a membraneimpermeant substance into a cell. (A) the substance is injected through a micropipette. (B) the cell membrane is made transiently permeable to the substance by disrupting the membrane structure with a brief but intense electric shock. (C) membranebounded vesicles are loaded with the desired substance and then induced to fuse with the target cells. Figure 364. Indirect immunocytochemistry. The method is very sensitive because the primary antibody is itself recognized by many molecules of the secondary antibody. The secondary antibody is covalently coupled to a marker molecule that makes it readily detectable. Commonly used marker molecules include fluorescein or rhodamine dyes, the enzyme horseradish peroxidase or colloidal gold spheres, and the enzymes alkaline phosphatase or peroxidase. Figure 365. Preparation of hybridomas that secrete monoclonal antibodies against a particular antigen (X). The selective growth medium used contains an inhibitor (aminopterin) that blocks the normal biosynthetic pathways by which nucleotides 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! 。 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。Chapter 3. Techniques in Cell Biology Preparatory observe put forward theoretics Design control tests Collect data Explain results Devise conclusion Refer to knowledge 從整個(gè)生命科學(xué)的發(fā)展趨勢(shì)看細(xì)胞 生物學(xué)方法 ? 分子水平 細(xì)胞水平 ? 結(jié)構(gòu)功能 細(xì)胞生命活動(dòng) ? 分析 綜合 ? 功能基因組學(xué)研究是細(xì)胞生物學(xué)研究的基礎(chǔ)與歸宿 （ 生命科學(xué)研究的核心問(wèn)題 ） Light Microscopy Figure 31. Resolving power. Sizes of cells and their ponents drawn on a logarithmic scale, indicating the range of objects that can be readily resolved by the naked eye and in the light and electron