Centre for Structural Systems Biology (CSSB), Hamburg, Germany.
Leibniz Institute of Virology (LIV), Hamburg, Germany.
Methods Mol Biol. 2024;2824:221-239. doi: 10.1007/978-1-0716-3926-9_15.
Cellular electron cryo-tomography (cryoET) produces high-resolution three-dimensional images of subcellular structures in a near-native frozen-hydrated state. These three-dimensional images are obtained by recording a series of two-dimensional tilt images on a transmission electron cryo-microscope that are subsequently back-projected to form a tomogram. Key to a successful experiment is however a high-quality sample. This chapter outlines a basic workflow for the preparation of cellular cryoET samples. It covers the preparation of infected cells on electron cryo-microscopy grids and the vitrification by plunge-freezing and clipping of grids into AutoGrid rims. It also provides a general overview of the workflow for thinning the vitrified cells by focused ion beam (FIB) milling. Although this book is dedicated to Rift Valley fever virus research, the present protocol may also be applied to any other research subject where high-resolution structural insight into intracellular processes is desired.
细胞电子冷冻断层成像（cryoET）以近天然的冷冻水合状态产生亚细胞结构的高分辨率三维图像。这些三维图像是通过在透射电子冷冻显微镜上记录一系列二维倾斜图像获得的，然后对这些图像进行反向投影以形成断层扫描图像。然而，成功实验的关键是高质量的样品。本章概述了细胞 cryoET 样品制备的基本工作流程。它涵盖了在电子冷冻显微镜网格上制备感染细胞以及通过无浸泡冷冻和将网格夹入 AutoGrid 边缘进行玻璃化的过程。它还提供了通过聚焦离子束（FIB）铣削对玻璃化细胞进行减薄的一般工作流程概述。尽管本书专门介绍裂谷热病毒研究，但本方案也可应用于任何其他需要对细胞内过程进行高分辨率结构洞察的研究课题。
