北京大学：《细胞生物学 Cell Science》课程教学资源（PPT课件讲稿）Electron microscopy

2. Electron microscopy Transmission Electron microscopy (TEMs) Scanning Electron microscopy(SE 1) Transmission Electron microscopy(TEMs) 1931 Ruska 1939 Siemens commercialisation, 1945 used in cells, OSO4 1948 Peale baker bio-section

2. Electron microscopy Transmission Electron microscopy (TEMs) Scanning Electron microscopy (SE 1) Transmission Electron microscopy (TEMs) 1931 Ruska 1939 Siemens commercialisation, 1945 used in cells, OsO4 1948 Peale Baker bio-section

电子显微镜的基本构造 Cathode(tungsten Electron gun nside Wehnelt First condenser Condenser condenser ens system 俯米 室品料 Pro ector lens 回中 VIewing screen 窗题 Photographic plate Figure A-21 A Transmission Electron Microscope (in camera chamber (a) A photograph and (b) a schematic diagram of a TEM

电子显微镜的基本构造

*EM structure: EM consist largely of (1)Acylindrical column (a) Cathode, tungsten wire filament, a source of electron (b) Electromagnet lens, 1000-250,000X magnification (c) Screen, photographic plate, or film 2)Vacuum system (3) Electronically control console prowerful suppler 104-105V The formation of an image in the TEM depends on the differential scattering of electron by parts of the specimen The later is proportional to the amount of matter present in that part, that is its mass thickness, which is a measure of the number of atoms per unit area and their atomic density

*EM structure:EM consist largely of (1) A cylindrical column (a) Cathode, tungsten wire filament, a source of electron (b) Electromagnet lens, 1000-250,000 X magnification (c) Screen, photographic plate, or film (2) Vacuum system (3) Electronically control console prowerful suppler 104 -105V The formation of an image in the TEM depends on the differential scattering of electron by parts of the specimen. The later is proportional to the amount of matter present in that part, that is its mass thickness, which is a measure of the number of atoms per unit area and their atomic density

光源(电子枪) 聚光镜 样品 描线圈 物镜 样品 显像管 一照印 探头 可观察的像 荧光板上的像 光学显微镜 透射电镜 扫描电镜 光学显微镜和电子显微镜的简单原理示意图

光源 电子枪(阴极 阳极 聚光镜 粉()磁聚光 标本 标本 物镜 V磁物镜 囹中间镜 中间象 中间象 目镜 粉器磁投影德 终象(眼或底片) 荧光屏成象

电镜与光镜光路图比较 回+回 Light microscope Electron microscope Figure 18.12 A comparison of the lens systems of a light and electron microscope(From w. Agar, Principles nd Practice of Electron Microscope Operat tion, Elsevier/ th-Holland, 1974)

电镜与光镜光路图比较

入=150V Work V:104-105Vat60,000,=0.05A theoretically D=0.61x 0.05/1x1=0.03 A Practically, resolution limit: 3-4 A, for Cellular structure 10-15 A

= 150/V Work V: 104—105 V at 60,000V,  =0.05 Å theoretically D = 0.61x 0.05 / 1x1= ~0.03 Å Practically, resolution limit: 3-4 Å, for Cellular structure 10-15 Å

电镜与光镜的比较 电子显微镜与光学显微镜的基本区别 分辨本领光源透镜真空成像原理 0.2mm 可见光 光学显微镜 可见光 利用样本对光的吸收形成明暗 反差和颜色变化 100nm 紫外光 玻璃透镜不要求真空 (波长约200mm) 电子显微镜接近0.1mm电子束 电磁透镜 33×10 利用样品对电子的散射和透射形 (波长 1.33×10°Pa成明暗反差

电镜与光镜的比较 电子显微镜与光学显微镜的基本区别 分辨本领 光 源 透 镜 真 空 成像原理 人眼 0.2mm 可见光 光学显微镜 200nm 可见光 ( 波 长 400 ～ 700nm) 利用样本对光的吸收形成明暗 反差和颜色变化 100nm 紫外光 （波长约 200nm） 玻璃透镜 不要求真空 电子显微镜 接近 0.1nm 电子束 ( 波 长 0.01 ～ 0.9nm) 电磁透镜 1.33×1 0-3～ 1.33×1 0-5P a 利用样品对电子的散射和透射形 成明暗反差

s Specimen preparation for TEM Standard method: Fixation: Glutaraldehyde( proteins cross-linking),osmium tetroxide(lipid), Dehydration(in alcohol) and embedding within epoxy plastic called epon Section: 0.02-0.1 u m, cut by glass or diamond Section mounting on grids and stain by electron dyes, which provide the mass thickness required to scatter the electron beam: uranyl acetate and lead citrate or be treated with metal-tagged antibody ect. Or a number of cytochemical procedures to detect some ei enzymes as acid phosphatase

* Specimen preparation for TEM Standard method: Fixation: Glutaraldehyde ( proteins cross-linking), osmium tetroxide (lipid), Dehydration (in alcohol) and embedding within epoxy plastic called Epon Section: 0.02-0.1μm, cut by glass or diamond Section mounting on grids and stain by electron dyes, which provide the mass thickness required to scatter the electron beam: uranyl acetate and lead citrate, or be treated with metal-tagged antibody ect. Or a number of cytochemical procedures to detect some enzymes as acid phosphatase

Tissue dissected out After washing, the tissue is and placed in fixing Tissue is now placed in Specimen dehydrated by placing it dilute solution of plastic in higher and higher concentrations of acetone or alcohol Specimen holder for microtome When the plastic is hard. the block is trimmed and is ady Tissue is placed in final bedding mixture and the polymerized Sections are cut on an ultramicrotome with a glass or diamond knife The sections are floated off the edge of the knife onto the surface of a water trough The sections are picked off th After the sections dry, they surface with a copper grid in the electron microscope