Morado Dustin R, Hu Bo, Liu Jun
Department of Pathology and Laboratory Medicine, University of Texas Health Science Center at Houston.
Department of Pathology and Laboratory Medicine, University of Texas Health Science Center at Houston;
J Vis Exp. 2016 Jan 30(107):e53608. doi: 10.3791/53608.
Cryo-electron tomography (Cryo-ET) is a powerful three-dimensional (3-D) imaging technique for visualizing macromolecular complexes in their native context at a molecular level. The technique involves initially preserving the sample in its native state by rapidly freezing the specimen in vitreous ice, then collecting a series of micrographs from different angles at high magnification, and finally computationally reconstructing a 3-D density map. The frozen-hydrated specimen is extremely sensitive to the electron beam and so micrographs are collected at very low electron doses to limit the radiation damage. As a result, the raw cryo-tomogram has a very low signal to noise ratio characterized by an intrinsically noisy image. To better visualize subjects of interest, conventional imaging analysis and sub-tomogram averaging in which sub-tomograms of the subject are extracted from the initial tomogram and aligned and averaged are utilized to improve both contrast and resolution. Large datasets of tilt-series are essential to understanding and resolving the complexes at different states, conditions, or mutations as well as obtaining a large enough collection of sub-tomograms for averaging and classification. Collecting and processing this data can be a major obstacle preventing further analysis. Here we describe a high-throughput cryo-ET protocol based on a computer-controlled 300kV cryo-electron microscope, a direct detection device (DDD) camera and a highly effective, semi-automated image-processing pipeline software wrapper library tomoauto developed in-house. This protocol has been effectively utilized to visualize the intact type III secretion system (T3SS) in Shigella flexneri minicells. It can be applicable to any project suitable for cryo-ET.
冷冻电子断层扫描（Cryo-ET）是一种强大的三维（3-D）成像技术，可在分子水平上可视化天然环境中的大分子复合物。该技术首先通过将标本快速冷冻在玻璃态冰中以保持其天然状态，然后在高倍率下从不同角度收集一系列显微照片，最后通过计算重建三维密度图。冷冻水合标本对电子束极为敏感，因此显微照片是在非常低的电子剂量下收集的，以限制辐射损伤。结果，原始的冷冻断层图具有非常低的信噪比，其特征是图像本质上有噪声。为了更好地可视化感兴趣的对象，采用了传统的成像分析和亚断层图平均方法，即从初始断层图中提取感兴趣对象的亚断层图，进行对齐和平均，以提高对比度和分辨率。大量的倾斜系列数据集对于理解和解析不同状态、条件或突变下的复合物以及获得足够大的亚断层图集合进行平均和分类至关重要。收集和处理这些数据可能是阻碍进一步分析的主要障碍。在这里，我们描述了一种基于计算机控制的300kV冷冻电子显微镜、直接检测设备（DDD）相机以及内部开发的高效、半自动图像处理管道软件包装库tomoauto的高通量冷冻电子断层扫描协议。该协议已有效地用于可视化福氏志贺氏菌微小细胞中的完整III型分泌系统（T3SS）。它适用于任何适合冷冻电子断层扫描的项目。
