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