细胞3,研究方法-2012.pdf

第三章 细胞生物学研究方法 Scientific Methods: Survival Skills for Young Investigators in Biological Research 主要学习内容 细胞形态结构的观察方法 细胞组分的分析方法 细胞培养、细胞工程与显微操作技术 第一节细胞形态结构的观察方法 1.光学显微镜技术 1.1 普通光学显微镜 A. 分辨率和放大倍数 分辨率是指光学成像系统 分辨两个相邻物点的能力。 介质折射率 样品对物镜镜口的张角 一般认为，人眼的分辨极限为0.2mm， 光学显微镜的分辨极限0.2μm，其放大 倍数为0.2mm/0.2μm=1000倍，这个倍 数被称为有效放大倍数 相差和微分干涉 显微镜 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 phase-contrast or a differential-interference-contrast microscope. 相差显微镜 （phase-contrast microscope ） 可将光程差或相位差转换成振幅差，从而提高各种结构 间的对比度, 可用于观察活细胞。 相差显微镜（phase-contrast microscope） 可将光程差或相位差转换成振幅差，从而提高各种 结构间的对比度, 可用于观察活细胞。 微分干涉显微镜 （differential-interference microscope ） 偏振光经合成后，使样品中厚度上的微小区别转化 明暗区别，增加了样品反差且具有立体感。适于研 究活细胞中较大的细胞器。 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 bright-field microscopy. The other images were obtained by techniques discussed in the text: (B) phase-contrast microscopy, (C) Nomarski differential-interference-contrast microscopy, and (D) dark- field microscopy. 1.2 荧 光 显 微 镜 应用 直接荧光标记技术 间接免疫荧光标记技术 荧光蛋白的应用 …… Fluorescent dyes. The structures of fluoresc