第二章细胞生物学方法技巧.pptVIP

第二章 细胞生物学研究方法;第一节 显微镜技术;（一）普通光学显微镜 ; 3.分辨率：指区分开两个质点间的最小距离。(肉眼、光学显微镜、电子显微镜的分辨率分别为0.2mm、0.2μm和0.2nm ) 光学显微镜的分辨力 R=0.61λ/N．A． 其中λ为入射光线波长； N．A．为镜口率 ＝ｎsinα/2， n=介质折射率； α=镜口角（样品对物镜镜口的张角） 光学显微镜理论放大倍数可以达到1500倍，但由于受到可见光光源衍射效应的限制，其有效放大倍数只能达到1000倍。;Preparation of specimen;1. 用于观察活体细胞； 2. 特殊之处： 环形光阑：位于光源与聚光器之间，作用是使透过聚光器的光线形成空心光锥，焦聚到标本上。 环形相板：在物镜中加了涂有氟化镁的相位板，可将直射光或衍射光的相位推迟1/4λ。 3. 原理 把光程差变成振幅差，提高了各种结构间的对比度，使其清晰可见。;相差显微镜光路图;莱卡相差显微镜（物镜与照明系统颠倒 ）;用途：适用于观察活细胞和活组织，是体外细胞和组织培养不可或缺的工具。;插入动态视频;DIC显微镜下的硅藻（伪彩色） ;（四）暗视野显微镜; ?????????????????????????????????????????????????????????????????????????????????????????? (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. ;（五）荧光显微镜;荧光显微镜照片 （微管呈绿色、微丝红色、核蓝色）;细胞形态观察、细胞内生化成分定性定量分析;（六）激光共聚焦扫描显微境 ;laser confocal scanning microscope, LCSM;（七）当代显微镜的发展趋势;二、电子显微镜技术;Scanning electron microscope（ SEM）;SEM LIGHT PATHWAY;Scanning electron micrograph of the stereocilia projecting from a hair cell in the inner ear of a bullfrog（牛蛙内耳毛发细胞） (A). For comparison, the same structure is shown by differential-interference-contrast light microscopy (B) and by thin-section electron microscopy (C).;Scanning electron micrograph of rat fibroblasts growing on the plastic surface of a tissue-culture dish. ;;Principal features of a light microscope, a transmission electron microscope, and a scanning electron microscope. These drawings emphasize the similarities of overall design. Whereas the lenses in the light microscope are made of glass, those in the electron microscope are magnetic coils. ;电镜与光镜的比较;2.光镜与电镜的主要区别;3.电镜的构造;Electron micrograph of a root-tip cell stained with osmium and other heavy metal ions. ;4.主要电镜制样技术;2）负染技术;;3）冷冻断裂和冰冻蚀刻电镜技术;Preparation of a metal-shadowed replica o