Schematic representation of the cryo-ET workflow. Vitrification. The sample type usually dictates the vitrification method. Samples that are thin enough to be directly investigated by EM are commonly vitrified by plunge-freezing into a cryogen (such as liquid ethane at less than −160°C) where sufficiently fast cooling speeds are reached (Dubochet et al., 1988). High-pressure freezing combines rapid temperature decrease with the application of high pressure, which lowers the melting point of water. This allows vitrification of samples up to 200 µm in thickness, but cryo-protectants are often needed (Studer et al., 2008). Thinning. Due to a strong electron–matter interaction, samples thicker than 1 µm are virtually opaque to electrons and require thinning at cryo-conditions. Although vitreous sectioning is currently used more often, focused ion beam milling (FIB) is an emerging method with a great promise for the future (see Box 2). Electron tomography. 3D images of vitrified samples are produced by tomographic imaging (see Box 1 for more information). Data analysis. Computational methods are used to improve the resolution of structures visualized in tomograms and to aid in their molecular identification. Cryo-correlative light microscopy is an optional procedure that facilitates identification and navigation to target features in cryo-ET.