南师大细胞生物学考研第3章+细胞生物学方法汇总.ppt

显微镜观察范围 肉眼观察范围 3D Image Reconstruction （三）流式细胞术与流式分选 流式细胞术 在流动的状态中检测细胞或颗粒的各项特性 特异性强，灵敏度高，速度快，并能实现多参数分析 可检测的样本种类多样：外周血，骨髓，细胞穿刺液，洗脱液，实体组织，培养细胞 流式分选 以流动状态的单个细胞的各项特性进行细胞分离 也叫作 Fluorescence-Activated Cell Sorting (FACS) 16 16 16 3 显微分光光度测定Spectrophotometry Use of the Spec 20 （二）元素分析电镜技术 电子束 非弹性散射 样品 X射线 非弹性散射电子失去的能量是一个特征值，用它成像便能反映出元素的性质. 能量损失谱成像技术(ESI)直观显示样品中某元素的分布 X射线也是一个特征值 X射线微区分析不直观显示元素分布 1、概念 2、流式工作原理 通过激光激发高速流动的单个细胞所携带的各种荧光素和荧光染料，并检测由此产生的散射光和荧光的发射光，通过各种光信号的强弱来反应细胞的各种特征。 Flow Cell Injector Tip Fluorescence signals Focused laser beam Sheath fluid Purdue University Cytometry Laboratories 3、流式分析的类型 ★荧光标识 Fluorescence-Activated Cell Sorting (FACS) ★机械力分选 Mechanical Sorting ★电荷分选 Electrostatic Sorting 488 nm laser + - Fluorescence Activated Cell Sorting Charged Plates Single cells sorted into test tubes FSC Sensor Fluorescence detector Mechanical Sorting: Takes place within a Flow cell When a sort decision “Green cell” has been made it is diverted into a ‘catcher’ tube either by moving the tube into the stream: Laser beam interrogates cells o o o o o o o o o o o o o o o or by applying an acoustic pulse to the stream to divert the cell into the tube. ooooo ooooo ooooo ooo o Hydrodynamic focusing takes place within a flow cell. o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o Electrostatic Sorting : Stream-in-air Laser interrogation and signal processing followed by sort decision : white sort right, blue sort left, green or black no sort. oooo oooo oooo left waste right Various collection devices can be attached : tubes, slides, multi-well plates. Hydrodynamic focusing in a nozzle vibrated by a transducer produces a stream breaking into droplets. o o o o o o o o o o o o o o o o o o o o o o o o o