细胞生物第二章研究方法.ppt

; 一.显微成像技术 二.细胞工程技术 三.细胞组分的分离技术 四.细胞组分的分析方法 ;一、光学显微镜;分辨率(resolution，R)：;Interference between light waves. When two light waves combine 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. ;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. ;;取材－固定－脱水－包埋－切片－脱蜡－复水－染色－脱水－透明－封片－观察;b. 荧光显微镜;Fluorescent dyes. ;Fruit fly (Drosophila ) embryo;c.激光共聚焦 扫描显微镜;Comparison of conventional and confocal fluorescence microscopy. These two micrographs are of the same intact gastrula-stage Drosophila embryo that has been stained with a fluorescent probe for actin filaments. ;;d.相差显微镜;Four types of light microscopy. (A) The image of a fibroblast（成纤维细胞） in culture obtained by common light microscopy (B) phase-contrast microscopy, (C) differential-interference-contrast microscopy(微分干涉差显微镜 ), and (D) dark-field microscopy.;E. Electronic image processing;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 0.2 nm (2 ?).;;光镜与电镜的基本区别;电镜技术的基本特点 1）电子束照明系统，包括电子枪、聚光镜。电子 束比可见光的波长短得多，提高了分辨力。 2）电磁透镜成像系统聚焦成像。 3）真空系统：电子运行中如遇到气体分子将会被 散射，所以电镜镜筒中要求高真空。 4）记录系统：电子像是人眼看不到的，因此要用 荧光屏来显示或感光胶片作记录。;;电镜种类;Electron micrograph of a root-tip cell stained with osmium（锇 ） and other heavy metal ions. ;Immunogold electron microscopy. ;1.超薄切片（40～50nm）。 2.固定→包埋→ 切片→ 染色等过程要求高。 3.重金属盐染色或喷镀，以形成不同程度的 反差，提高分辨力。 4.负染技术 5.冰冻断裂和冰冻蚀刻技术;Specimen Preparation for Electron Microscopy;Chemical fixation; ?????????????????????????????????????????Diagram of the copper grid used to support the thin sections of a specimen in the transmission electron micros