Preparation of Bunyavirus-Infected Cells for Electron Cryo-Tomography

• Published in: Methods in molecular biology (Clifton, N.J.) Vol. 2824; p. 221
• Main Authors: Ott, Fanny, Jönsson, Märit-Runa, Grünewald, Kay, Hellert, Jan
• Format: Journal Article
• Language: English
• Published: United States 2024
• Subjects: Animals; Cryoelectron Microscopy - methods; Electron Microscope Tomography - methods; Humans; Imaging, Three-Dimensional - methods; Rift Valley fever virus - ultrastructure; Vitrification; Plunge-freezing; Cell culture; Focused ion beam milling; Virus infection; Electron cryo-microscopy; Vitrification
• ISSN: 1940-6029
• 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.