20210319-冷冻电镜技术.pdfVIP

冷冻电镜数据的收集和监控 常圣海 冷冻电镜中心（ CCEM ） 浙江大学 Outlines 1. 冷冻电镜数据收集的相关设定--SerialEM 数据格式的选择 Super vs Counting mode Multishot 2. 冷冻电镜数据的监控--Focus 1. Basic knowledge and related principles of cryoEM 1.1 why EM? 1.2 The principle of cryo-Sample preparation 1.3 The principle of 2D crystal diffraction 1.4 The principle of SPA 1.1 Why EM? Abbe Limit ： δ=0.61λ/(nSinα)， Electron wavelengths (λ) 0.0037nm at 100kV 0.0025nm at 200kV 0.0020nm at 300kV The limitation of EM for bio-sample High vacuum Electrons interact much more Low transmission strongly with matter capacity High radiation damage The rules of EM sample preparation •The thickness of the sample should be less than 500nm •The sample should be stable at high vacuum •The sample should not be sensitive to electron radiation Conventional methods for EM sample preparation conventional fixation embeding and section negative staining 1.2 Vitreous ice Cryo-EM specimen preparation is creating a well-preserved sample in a thin, vitreous ice Doubochet and his cryo-virus Low dose imaging leads to low contrast 是 vs 足 1.3 2D crystal diffraction 是 vs 足 是 vs 足 是 vs 足 是 vs 足