复旦大学细胞生物学课件03细胞生物学技术

1、Chapter 3Techniques in Cell Biology 普通光学显微镜（A）和荧光显微镜(B)的光路图。 1.The Light MicroscopyA. Preparation of specimen:B. Resolution and magnification The resolving power of a microscopecan be defined in terms of the ability to see two neighboring points in the visual field as distinct entities.N.A. : Numeri

2、cal aperture Limit of resolution of the lightmicroscope = 0.2um (200nm)Max. useful magnification: 500-1000 ?N.AFluorescence Microscopy Direct immunofluorescence technique Fluorochrome: Such as rhodamine or fluoresceinC. Special Light MicroscopesIndirect immunofluorescence lebeled Tchnique. To study

3、the location of a specific protein within the cell by using of fluorescent antibody (antigen-antibody couple).GFP can be used to study dynamic processes as they occur in a living cell. Confocal Scanning Light Microscopy 激光扫描共焦显微镜的原理图 A.激光束（光源）经双色镜反射后，通过物镜汇聚到样品某一焦点；B.从焦点发射的荧光(样品一般须经免疫荧光标记)经透镜汇聚成像，被检测

4、器检出；C. 通过样品其它部位的激光即激光发出的荧光不会聚焦成像，因而检测器不能检出。 In the late 1950s M.Minsky of MIT免疫荧光技术（A）和激光共焦显微镜技术(B)的比较 Phase-contrast microscope 相差显微镜 Differential-interference microscope微分干涉显微镜A comparison of yeast cells that were growing on the vaginal epithelium seen with different types of LM. Fluorescence Reso

5、nance Energy Transfer，FRET FRET技术是检测活体中生物大分子纳米级距离和纳米级距离变化的有力工具，可以检测某一细胞中两个蛋白是否存在直接的相互作用 荧光共振能量转移原理图:供体蛋白与受体蛋白无相互作用（A）和有相互作用（B）时的荧光变化(距离在510nm的范围内 )。FRET效率反映了体内两种蛋白是否直接相互作用及作用的强弱 Fluorescence Recovery After Photobleaching，FRAP 荧光漂白恢复原理: 利用高能量激光束的照射使特定的区域的荧光发生不可逆的淬灭，通过非漂白区的荧光标记分子在膜上或胞浆中运动至光漂白区来完成光漂白区荧

6、光的恢复。FRAP 可以用于解决活细胞内包括蛋白定位，动力学以及与其他成分相互作用等一系列问题。 Video-enhance(contrast) microscopyObserving living specimens;Greatly increase the contrast of an image so that very small objects become visible.分辨本领光 源透 镜真 空成像原理光学显微镜200nm可见光 (波长400700nm)玻璃透镜不要求真空利用样本对光的吸收形成明暗反差和颜色变化电子显微镜 接近0.1nm电子束(波长0.010.9nm)电磁透镜1

7、.3310-31.3310-5Pa利用样品对电子的散射和透射形成明暗反差电子显微镜与光学显微镜的基本区别 The resolving power for LM;The resolving ability(0.2nm) and resolving power(5nm) for TEM2. Transmission Electron MicroscopyA. The comparison of the lens systems of LM and TEM电子显微镜光成像原理 A.透射电子显微镜剖面图。B. 透射电子显微镜电子成像原理图。 B. Specimen Preparation for El

8、ectron MicroscopyThin Sectioning for TEMThe wax sections: 5um;The Plastic ultrathin-sections for TEM: 50-100nmSections of LM: 5um;Sections of TEM: 0.1 msB. Specimen Preparation for Electron Microscopy固定：锇酸、戊二醛包埋：环氧树脂切片：玻璃、钻石作为切片刀材料， 载网：铜网或镍网染色：重金属盐染色增加明暗反差 锇酸：脂质；铅盐：蛋白质；醋酸铀：核酸Negative staining and me

9、tal-shadowing（金属喷涂）Metal Shadowing allows surface features to be examined at high resolution by TEMExamples of negtively stained and metal-shadowed specimens.Electron micrographs of a tobacco rattle virus after negtive staining with potassium phosphotungstate(a) （磷钨酸钾）or shadow casting with chromium

10、(b)（铬）.Freeze Etching Fracture 冷冻蚀刻Freeze Fracture and Etching Replication冷冻断裂蚀刻复型quick freeze deep etching3. Scanning electron microscope (SEM) Images of surfaces can be obtained by SEM;Critical-point drying;Range: 15 150,000 Xs. Resolution: 5nm 4. The Scanning probe microscope( SPM) Scanning tunne

11、ling microscope(STM)Including: Scanning tunneling microscope (STM), Atom force microscope (AFM), Magnetic force microscope (MFM), Friction force microscope (FFM) STM的主要特点：（1）具有原子尺度的高分辨本领，侧分辨率为0.10.2nm，纵分辨率可达0.001nm；（2）可以在真空、大气、液体（接近于生理环境的离子强度）等多种条件下工作，这一点在生物学领域的研究中尤其重要；（3）非破坏性测量。因为扫描时不接触样品，又没有高能电子束轰

12、击，基本上可避免样品的形变。用途：纳米生物学研究领域中的重要工具.5.The Fractionation and analysis for Cell s contentsA.The technique of differential centrifugationStep-by-step procedure for the purification of organelles by differential centrifugation.S=(dx/dt)/2x =110-13sec.用差速离心分离各细胞组分。在细胞匀浆物中，较小的细胞组分需要更大的离心力才能使其沉淀。 1000g, 10min

13、, 20000g, 20min， 高速： 80,000g, 1hr, 150，000g, 3hr。 B. Subsequent purification by Density-Gradient Equilibrium CentrifugationC. Isolation, purification, and fractionation of proteins: Selective Precipitation (ammonium sulfate) Liquid Column Chromatography （柱层析） Ion-exchange Chromatography Gel Filtrati

14、on Chromatography 又称排阻层析或分子筛方法：交联的聚糖(如葡聚糖或琼脂糖) Affinity Chromatography Polyacrylamide Gel ElectrophoresisD. Determining Protein-Protein InteractionImmunoblot: Western-BlotE.Localization of a specific protein by immuno-electron microscopyImmuno-gold electron microscopyF. Nucleic acid hybridizationDet

15、ermining the location of specific DNA fragments in a gel by a Southern blotFluorescent in situ Hybridization (FISH) Isotope-labeled probe or Biotin-labeled probe G. Ezymatic Amplification of DNA by PCR用原位杂交技术显示Z13基因在受精后1天的斑马鱼胚胎的体节、眼和松果体中的表达（箭号）。(张博惠赠)鸭瘟病毒感染鸡胚成纤维细胞24小时后，用3H尿嘧啶核苷脉冲标记10分钟的电镜放射自显影图片。曝光时

16、间90天，核仁（Nu）和细胞核的其它部位均有RNA合成。SG： 银颗粒； N： 细胞核； Nu： 核仁； C： 细胞质。（丁明孝，翟中和） 2022/9/1048Biochips)Gene Microarray (6. Protein structure determinationX-ray Crystallography A major technique that has been used to discover the three-dimensional structure of protein molecules, at atomic resolution. B. Nuclear M

17、agnetic Resonance (NMR) SpectroscopyNMR technique has been used to study the structure of small protein(MW200KD) or protein domains.7. Flow Cytometry流式细胞分选仪工作原理。 （A）当含有单个细胞的液滴通过激光束时，带有不同荧光的细胞所在的液滴被充上正电荷、负电荷或不被充电。因带有不同表面标志的细胞所带的电荷的不同，当液滴通过高压偏转板时，液滴发生偏转，从而达到将细胞分选的目的。（B）显示流式细胞仪分析处在不同时相的Hela细胞的实验结果（2N：G

18、1期，2N4N：S期，4N：G2/M期） 8. Autoradiograph of LM and TEM9.The The Cell the s cultures and Cell s engineeringPrimary culture cell and subculture cell: Cell strain and Cell lineHeLa, BHK21(baby hamster kidney), CHO(chinese hamster ovary);Two types of morphes: fibroblast like cell and epithelial like cell;

19、Protoplast culture and anther culture在非洲爪蟾卵细胞提取物非细胞体系中加入噬菌体DNA诱导形成细胞核（N）（张博，翟中和等）B. Cell-free systemMonoclone antibodyCesar Milstein and Georges Kohler1975, in Cambridge developing: The hybridomas techniques.HAT( Hypoxanthin,aminopterin, and thymidine) selective medium: The cell with HGPRT(次黄嘌呤鸟嘌呤磷酸

20、核糖基转移酶) growing;The The cell lacking HGPRT don t growing. C. Cell s engineering Cell fusion and cell hybridization2022/9/1061Monoclonal antibody technology小鼠脾（B淋巴细胞）X骨髓瘤细胞 细胞融合HAT培养基H：hypoxanthine 次黄嘌呤A:aminopterin 氨基蝶呤：阻断核酸合成主通道T:thymine 胸腺嘧啶（突变肿瘤细胞：无胸腺嘧啶核苷激酶(TK,合成DNA旁路酶)和 磷酸核糖转移酶(HGPRT,合成RNA旁路酶） D

21、. The technique for the take apart and gather up of cell, and microscope manipulation Preparation and reform of karyoplast and cytoplastTransgenic animals and plantsTransgenic mice10 weeks44g and 29g The Ralph Brinster(, the University of Pennsylvania) and Richard Palmiterand (University of Washingt

22、on ) 1981 2022/9/1064Knockout Mice The technique used to generate knockout mice was developed in the late 1980s by Marlo Capecchi at the University of Utah. 实验分三步 构建重组体； 转基因敲除； 筛选。Knockout miceMario Capecchi (Late 1980s) (University of Utah)embryonic stem cells in inner cell mass as target cells1/10

23、4 cells undergo a process of homologous recombination. Model organisms of study on cell biology 大肠杆菌与操纵子学说的建立及现代分子生物学的发展；豌豆和果蝇与遗传学定律的发现；酵母和海胆与对细胞周期调控机制的认识；线虫与对细胞凋亡机制的揭示；小鼠与哺乳动物功能基因组学的研究等等。2022/9/1067Exogenous and Endogenous RNAiLiposome-mediated Transfection2022/9/1068Systems Biology of the CellCurrently, biologists tend to study the cell structure and behaviors as the ultimate objective to systemically understand the effect of the molecules on the whole cell, and the effect of the individual cell on the whole organism as well.cytomics 2022/9/10