《分子细胞生物学》 Chapter 3 Techniques in Cell Biolog

Chapter 3. Techniques in Cell Biology Preparatory observe put forward theoretics Design control tests Refer to knowledge Collect data Explain results Devise conclusion

Chapter 3. Techniques in Cell Biology Preparatory observe put forward theoretics Design control tests Collect data Explain results Devise conclusion Refer to knowledge

从整个生命科学的发展趋势看细胞 生物学方法 分子水平 细胞水平 结构功能 细胞生命活动 分析 综合 功能基因组学研究是细胞生物学研究的 基础与归宿 (生命科学研究的核心问题)

从整个生命科学的发展趋势看细胞 生物学方法 • 分子水平 细胞水平 • 结构功能 细胞生命活动 • 分析 综合 • 功能基因组学研究是细胞生物学研究的 基础与归宿 （生命科学研究的核心问题）

细胞生物学研究方法 显微观篝 细胞培养 分子生物学技术 组纵学找术 胚胎学技术 生物化学找术 物理学技术

IThe light microscopy Ocular (eyepiece Remagnifies the image formed by the objectve lens Ocular lens Body tube Transmits the image from the ob- Line of vision jective lens to the ocular Path of light Objective lenses Primary Body tube lenses that magnify the Obiective Stage Holds the microscope slide in postion Condenser Focuses Condenser ragm Controis the amount entering the condenser Illuminator. Coarse focusing knob Luminator Light source source of Base allum nation Fine focusing knob (a)Principal parts and functions (b) The path of light(bottom to top) Figure A-6 The Compound Light Microscope. (a)A compound light microscope. (b)The path of light through the compound microscope

1.The Light Microscopy

A. Resolution and magnification animal Figure 3-1. Resolving power. Sizes 10 um of cells and their components drawn on a logarithmIc scale, indicating the range of objects that can be readily resolved by the naked eye and in the light and electron 100mm virus microscopes. The following units of length are commonly employed in globular microscopy: um(micrometer)=10 protein 6 mnm(nanometer)=10-9m A (Angstrom unit)=10-10 m molecule atom 0.1nr

Figure 3-1. Resolving power. Sizes of cells and their components drawn on a logarithmic scale, indicating the range of objects that can be readily resolved by the naked eye and in the light and electron microscopes. The following units of length are commonly employed in microscopy: µm (micrometer) = 10- 6 m nm (nanometer) = 10-9 m Å (Ångström unit) = 10-10 m

TWO WAVES IN PHASE TWO WAVES OUT OF PHASE A个个0000 dim bright Figure 3-2. Interference between light waves. When two light waves combine in phase the amplitude of the resultant wave is larger and the brightness is increased. Two light waves that are out of phase partially cancel each other and produce a wave whose amplitude, and therefore brightness, is decreased

Figure 3-2. Interference between light waves. When two light waves combine in phase, the amplitude of the resultant wave is larger and the brightness is increased. Two light waves that are out of phase partially cancel each other and produce a wave whose amplitude, and therefore brightness, is decreased

Figure 3-3. Edge effects. The interference effects observed at high magnification when light passes the edges of a solid object placed between the light source and the observer

Figure 3-3. Edge effects. The interference effects observed at high magnification when light passes the edges of a solid object placed between the light source and the observer

LENSES RESOLUTION: the resolving power of the microscope depends on the width of the cone of illumination and therefore on both the condenser and the objective lens. It is IMAGE calculated using the formula the objective lens resolution 0.612. fsin B collects a cone of light rays to create where specimen an image e- half the angular width of the cone of rays collected by the objective lens the condenser lens from a typical point in the specimen focuses a cone of (since the maximum width is 180% light rays onto sin B has a maximum value of 1) each point of the r,. the refractive index of the medium specimen usually air or oil) separating the LIGHT specimen from the objective and condenser lenses i- the wavelength of light used (for white light, a figure of 0. 53 um is commonly ssumed) NUMERICAL APERTURE. r sin f in the aperture, the greater the resolution and the equation above is called the numerical aperture brighter the image (brightness is important in of the lens (NA) and is a function of its light- fluorescence microscopy). However, this advan- collecting ability For dry lenses this cannot be tage is obtained at the expense of very short more than 1, but for oil-immersion lenses it can working distances and a very small depth of field be as high as 1. 4. The higher the numerical Figure 3-4. Numerical aperture. The path of light rays passing through a transparent specimen in a microscope, illustrating the concept of numerical aperture and its relation to the limit of resolution

Figure 3-4. Numerical aperture. The path of light rays passing through a transparent specimen in a microscope, illustrating the concept of numerical aperture and its relation to the limit of resolution

specimen embedded in wax or resin steel blade B. Preparation of specimen ribbon of sections on glass slide. stained and mounted under a cover shp 游踐路馒淤激哥骱瑟 bjective lens condenser Figure 3-5. Making tissue sections. How an embedded tissue is sectioned with a microtome in preparation for examination in the light microscope

Figure 3-5. Making tissue sections. How an embedded tissue is sectioned with a microtome in preparation for examination in the light microscope. B. Preparation of specimen