【正文】 Chapter 3. Techniques in Cell Biology Preparatory observe put forward theoretics Design control tests Collect data Explain results Devise conclusion Refer to knowledge 從整個生命科學(xué)的發(fā)展趨勢看細胞 生物學(xué)方法 ? 分子水平 細胞水平 ? 結(jié)構(gòu)功能 細胞生命活動 ? 分析 綜合 ? 功能基因組學(xué)研究是細胞生物學(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 microscopes. The following units of length are monly employed in microscopy: 181。m (micrometer) = 106 m nm (nanometer) = 109 m 197。 (197。ngstr246。m unit) = 1010 m Figure 32. Interference between light waves. When two light waves bine in phase, the amplitude of the resultant wave is larger and the brightness is increased. Two light waves that are out of phase partially cancel each other and produce a wave whose amplitude, and therefore brightness, is decreased. Figure 33. Edge effects. The interference effects observed at high magnification when light passes the edges of a solid object placed between the light source and the observer. Figure 34. Numerical aperture. The path of light rays passing through a transparent specimen in a microscope, illustrating the concept of numerical aperture and its relation to the limit of resolution. Figure 35. Making tissue sections. How an embedded tissue is sectioned with a microtome in preparation for examination in the light microscope. B. Preparation of specimen Figure 37. The optical system of a modern fluorescence microscope. A filter set consists of two barrier filters (1 and 3) and a dichroic (beamsplitting) mirror (2). In this example the filter set for detection of the fluorescent molecule fluorescein is shown. C. Fluorescence Microscopy Figure 38. Fluorescent dyes. The structures of fluorescein and tetramethylrhodamine, two dyes that are monly used for fluorescence microscopy. Fluorescein emits green light, whereas the rhodamine dye emits red light. Figure 39. Fluorescence microscopy. Micrographs of a portion of the surface of an early Drosophila embryo in which the microtubules have been labeled with an antibody coupled to fluorescein (left panel) and the actin filaments have been labeled with an antibody coupled to rhodamine (middle panel). In addition, the chromosomes have been labeled with a third dye that fluoresces only when it binds to DNA (right panel). At this stage, all the nuclei of the embryo share a mon cytoplasm, and they are in the metaphase stage of mitosis. The three micrographs were taken of the same region of a fixed embryo using three different filter sets in the fluorescence microscope. Figure 310. Two ways to obtain contrast in light microscopy. The stained portions of the cell in (A) reduce the amplitude of light waves of particular wavelengths passing through them. A colored image of the cell is thereby obtained that is visible in the ordinary way. Light passing through the unstained, living cell (B) undergoes very little change in amplitude, and the structural details cannot be seen even if the image is highly magnified. The phase of the light, however, is altered by its passage through the cell, and small phase differences can be made visible by exploiting interference effects using a phasecontrast or a differentialinterferencecontrast microscope. D. Phasecontrast or a differentialinterference contrast microscope Figure 311. Four types of light microscopy. (A) The image of a fibroblast in culture obtained by the simple transmission of light through the cell, a technique known as brightfield microscopy. The other images were obtained by techniques discussed in the text: (B) phasecontrast microscopy, (C) Nomarski differentialinterferencecontrast microscopy, and (D) darkfield microscopy. Figure 312. Extending the limits of detection. Lightmicroscope images of unstained microtubules that have been visualized by differentialinterferencecontrast microscopy followed by electronic image processing. (A) The original unprocessed image. (B) The final result of an electronic process that greatly enhances contrast and reduces noise. (Courtesy of Bruce Schnapp.) E. Electronic image processing Videoenhance(contrast) microscopy ?Observing living specimens。 ?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 i