激光共聚焦PPT.ppt

时间序列 时间序列记录（真实时间） 在线比率 内/外触发（4内4外） 荧光漂白程序 事件标记 像素时间 感兴趣区域/线的荧光强度平均值（在线或离线） 最大时间分辨率： 幅：5 fps by 512x512 或 77 fps by 512x32 线（ 512像素）： ~ 2600 / sec. (0.38 ms) 速度级别：13X2 ? Application Time - Lapse 共聚焦经典应用 Live Cell Imaging Emission Fingerprinting Having the right equipment - live cell imaging can by a colorful adventure! Specimen: cell culture, CFP/GFP/YFP fused each to histone 2B Imaging: 16h time series acquisition of lambda / z stack Image processing: Linear Unmixing, maximum projection Courtsey Prof. Saiwaki, Japan 5D Imaging: x,y,z,l, t Series Application Ion Imaging Ion imaging What is ion imaging? Ion imaging is the visualization of free ions (e.g. Ca) in living cells or tissues using fluorescent probes that change their emission properties in dependence of the free ion concentration. What is it good for? Using ion-sensitive dyes one can study the dynamic changes (fast and slow) in ion concentration that are indicative for inter- and intracellular cell communication. Ca transients in salivary glands of the blowfly (fluo-4, 5 frames per second) B. Zimmermann, University of Potsdam, Germany ? Online imaging of the calcium sensor Fluo-4: Intercellular calcium wave induced by a hormone in a salivary gland of a fly Online image display as F/F0 F0 automatically calculated from 10 pre-stimulus images during acquisition 3.5 0.8 F/F0 ? Quantitative: Enhanced Physiology I Application Colocalization The new colocalization dialog Scattergram, image display and data table are interactively linked Scattergram Data table Image display Tools Quantitative Colocalization Analysis 主要应用 一、静态检测 1.高分辨率共聚焦图片 有效抑制同一焦平面上非测量点的杂散荧光及来自样品中非焦平面的荧光，从而获得普通光镜无法达到的分辨力；逐层获得高反差、高分辨率、高灵敏度的二维光学横断面图像，获得无损伤的系列光学切片 2. 三维重建 经计算机图像处理三维重建软件重组，可得到标本的三维立体结构，揭示亚细胞结构的空间关系 3.定性、定量分析 对单细胞或细胞群的溶酶体、线粒体、内质网、细胞骨架、结构性蛋白质、DNA、RNA、酶和受体分子等细胞结构的含量、组分及分布进行定性、定量及定位测定。另外可对细胞的面积、细胞周长等参数进行自动测定。 二、动态监测 1、活体细胞或组织功能的实时动态检测