Chapter 3 Techniques in Cell Biology Preparatory observation Hypothesis Design controls Refer to literature Collect data Methodology Interpret results Draw conclusions
Chapter 3 Techniques in Cell Biology
IThe Light microscopy 目镜 目镜 3.第二组阻断滤片 物镜 2.反光镜 聚光镜 1,激发光阻断滤片 物镜 光源 普通光学显微镜(A)和荧光显微镜(B的光路图
普通光学显微镜（A）和荧光显微镜(B)的光路图。 1.The Light Microscopy
A. Preparation of specimen Specimen embedded Microtome arm in paraffin wax or plastic resin Metal or glass blade Ribbon of thin Ribbon of sections on glass slide, stained and mounted under a cover sIp c Figure A-18 Sectioning with a Microtome. The fixed specimen is embedded in paraffin wax or plastic resin and mounted on the arm of the microtome. As the arm moves up and down through a circular arc, the blade cuts successive sections. The sections adhere to each other forming a ribbon of thin sections that can be mounted on a glass slide stained, and protected with a coverslip
A. Preparation of specimen:
B. Resolution and magnification The resolving power of a microscope can be defined in terms of the ability to see two neigh boring points in the visual field as distinct entities 隨镜 图像 N.A.: Numerical aperture Limit of resolution of the light microscope=0.2um(200nm) 样品 Max useful magnification: 500-10002N.A 0.61 ·S|na 1/2
B. Resolution and magnification The resolving power of a microscope can be defined in terms of the ability to see two neighboring points in the visual field as distinct entities. N.A. : Numerical aperture Limit of resolution of the light microscope = 0.2um (200nm) Max. useful magnification: 500-1000 ?N.A
C Special light Microscopes Fluorescence Microscopy Direct immunofluorescence technique Fluorochrome: Such as rhodamine or fluorescein Fluorescence microscopy Fluorescence microscopy Chromosome Actin fibers in a fibroblast
Fluorescence Microscopy ❖ Direct immunofluorescence technique ❖ Fluorochrome: Such as rhodamine or fluorescein C. Special Light Microscopes
Visualizing intracellular Ca2+ concentration by the fluorescent indicator fura-2 in a Purkinje cell 100um
Visualizing intracellular Ca2+ change by the fluorescent indicator fura-2 15 s after sperm added 0080 3 min 32s ...息 ca2+】1
ndirect immunofluorescence labeled Tchnique To study 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. Dynamics of GFP tagging: follow the movement of spliceosome in the nucleus in a living cell
❖Indirect immunofluorescence lebeled Tchnique. To study 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
Indirect immunocytochemistry primary antibody: secondary antibodies: rabbit antibody marker- coupled antibodies directed against directed against rabbit antigen A antibodies 4 marker immobilized antigen A Markers: fluorescence dyes, horseradish peroxidase, alkaline phosphatase, colloidal gold
Confocal Scanning Light Microscopy In the late 1950s M. minsky of mIT 共焦小孔 双色镜 物镜 样品 焦点 (A) (B) B C 免疫荧光技术(A)和激光共焦显微镜技术(B的比较 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
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