武汉大学：《细胞生物学 Cell Biology》课程教学资源（PPT课件讲稿）第三章 细胞生物学研究方法

第三章细胞生物学研究方法 第一节细胞形态结构的观察方法

第三章 细胞生物学研究方法 第一节 细胞形态结构的观察方法

光学显微镜技术 Icm Imm 100um 10um lum 100nm 10nm Inm 0.Inm ←光学显微镜 电子显微镜 扫描隧道显微镜 图3-1各种细胞及其组分的大小在对数刻度尺上的分布图 及几种显微镜的分辨范围

一 光学显微镜技术 1cm 1mm 100μm 10μm 1μm 100nm 10nm 1nm 0.1nm 光学显微镜 电子显微镜 扫描隧道显微镜 图 3--1 各种细胞及其组分的大小在对数刻度尺上的分布图 及几种显微镜的分辨范围

(一)普通复式光学显微镜技术 光学显微镜的组成主 ring 要分三部分 (1)光学放大系统; 2)照明系统; lenses (3)机械和支架系统。即m Mechanig 普通光镜的最大分辨率neos 是0.2微米 knob Slag adjusment Condenser c。ndsn:r Bright ness Daylight Parts of a Nikon compound light cord fliter ONOFF microscope

（一）普通复式光学显微镜技术 光学显微镜的组成主 要分三部分: （1）光学放大系统； （2）照明系统； （3）机械和支架系统。 普通光镜的最大分辨率 是0.2微米。 Parts of a Nikon compound light microscope

lo noirmni1 ei Cells linin blood vassel Blood vessel with red blood cells Connective tissue cells Hepatocyte [liver cell L100g A Figure 4-10 A light-microscopic view of a section of buman liver, stained with the dyes cosin and methylenc blue. Note that at least four diffcrcnt cell types arc distinguishable by this staining protocol, Courtesy of P. G. Aitken/Biophoto associate

(二)荧光显微镜技术 特异蛋白质等生物大分子定性定位的最有力的工具。 Barrier filter Exciter matic A Figure 4-12 The epi-fluorescence microscopc. Light from a multiwavelength source moves through an exciter Filter, which allows only the desired wavelength of exciting radia- tion to pass. This radiation is reflected by the dichromatic filer and focused by the objective lens onto the sample; luo. Objective lens rescent molecules in the sample arc then excited to emit light oresce)at a specific and longer wavelength focuscd by the objective lens; most of it passes through che Specimen dichromatic filter and is not reflected. A final barrier filter blocks any residual light with the frequcncy of the exciting

（二）荧光显微镜技术 特异蛋白质等生物大分子定性定位的最有力的工具

s 4 A Figure 4-13 Distribution of actin in a cultured fibroblast A Figure 4-14 Fluorescence inicrogral aph of che endoplasmic cell. A fixed human skin fibroblast was permeabilized with a reticulum(ER)in a flattened rcgion of a living monkey kid- detergent and stained with a fluorescene antiactin antibody. ney epithelial cell, The ER is a set of membrane fibers that fibers within it. Courtesy of E. Lazarides. eat larger, nonreticular fluorescent structures are mitochon o This fluorescence micrograph of a cell shows the long actin fuse with each other to form a network, or reticulum. The dria(M).[Scc C. Lee and L. B. Chen, 1988. Cel! 44: 37-46. Courtesy of L. B. Clen. ouiat2i788-1r dunhe tin

三)激光共焦点扫描显微 镜技术 比普通荧光显微镜的分辨率 → Phase pl 提高14--1.7倍。 obstructed light objective lens 通过”光学切片”观察较厚样 品的内部结构。 Condensor lons (四)相差和微分干涉显微 镜技术 相差显微镜的样品不需染色 可以观察活细胞,甚至研究细胞 核、线粒体等细胞器的动态。 Incident light pular amilus (ring) of light on 微分千涉显微镜更适于研究活 细胞中较大的细胞器。 ves. This produces an of a region of che sample depends on the fractive index of ch

（三）激光共焦点扫描显微 镜技术 比普通荧光显微镜的分辨率 提高1.4---1.7倍。 通过”光学切片”观察较厚样 品的内部结构。 （四）相差和微分干涉显微 镜技术 相差显微镜的样品不需染色, 可以观察活细胞，甚至研究细胞 核、线粒体等细胞器的动态。 微分干涉显微镜更适于研究活 细胞中较大的细胞器

Tungsten filament 二电子显微镜技术 Beam of electrons Condenser lens (一)电子显微镜的 基本知识 objective lenses 1.电子显微镜与光学显微 Focal plane of 镜的基本区别 objective len 2电子显微镜的分辨本领 与有效放大倍数 Viewing screen 3电子显微镜的基本构造 A Figure 4-23 The optical path in a transmission electron tungsten filament is focused onto the specimen plane by the magnetic condenser lens. The electrons passing through the specimen are focused by a series of objective and projector lenses to form a magnified image of the specimen on a fluo rescent viewing screen or a piece of photographic filn. The entire columm, from the electron generator to the screen, is maintained at a very high vad ulm

二 电子显微镜技术 ◼ （一）电子显微镜的 基本知识 ◼ 1.电子显微镜与光学显微 镜的基本区别 ◼ 2.电子显微镜的分辨本领 与有效放大倍数 ◼ 3.电子显微镜的基本构造

Ultramicrotome (二)主要电镜技术介绍 trough 数rd 超薄切片技术 ■2.负染色技术 ly for viewing ■3.冷冻断裂和冷冻蚀刻 Copper grid 电镜技术 ▲ Figure4-24 Preparation of a sample of tissue for trans ■4.电镜三维重构技术 nission electron microscopy. The tissue is dissected, cut into small cubes, and plunged into a fixing solution that cross links and immobilizes proteins.(Ghtaraldchydc is frcquently ■5.扫描电镜技术 used; ostium tetroxide, another fixing substance, stains in- tracellular membranes and certain macromolecules. )The sample is dehydrated by placing it in successively more con CO临界点干燥法 centraced solutions of alcohol or acetone: it is then immersed in a solution of plastic embedding medium and put in an oven. Heat causes the solution to polymerize into a hard plastic black, which is trined; sections less than 0. 1um thick are then cur with an ultramicrotome, a fine slicing in off the blade edge onto the surface of water in a trough. A opper grid coated with carbon or some other matcrial is 5 used to pick up the sections, which are then dried

（二）主要电镜技术介绍 ◼ 1. 超薄切片技术 ◼ 2. 负染色技术 ◼ 3. 冷冻断裂和冷冻蚀刻 电镜技术 ◼ 4. 电镜三维重构技术 ◼ 5. 扫描电镜技术 ◼ CO2临界点干燥法

Nucleus uclear pores Golgi vesicle Inner nucl Outcr nuclear Nuclear pores Rough ER micrograph of a thin section of an Arabidopsis(plant, A Figure 4-34 Views of the nuclear envelope. (a)Electre showing the porcs between the nuclear membranes. ' The rough endoplasmic reticulum and stacks of Golgi vesicles are lc.(b) A freeze-fractured be filled with ular sub stance, Part (a) courtesy of Biopboto Associates Myron C,E Ledbetter/Brookhaven National Laboratory; part(b)courtesy of D. Brar