Samples containing biological material need to be fixed prior to EM imaging. Chemical fixation works well, but induces many artefacts, and strongly disturbs the ultrastructure of the samples. Cryofixation is a fixation method that involves freezing the sample so quickly that even water molecules don’t have the time to form ice-crystals, thereby optimally preserving the sample in a near-native state. Various techniques exist, such as plunge-freezing, jet-freezing and high-pressure freezing. The most common method of cryofixation is plunge-freezing, where the sample is plunged rapidly in a cryogen such as liquid ethane cooled by liquid nitrogen. We have two systems available, the Thermo Scientific Vitrobot and the Leica EM GP2. Plunge-freezing is suited for thin samples, such as single-proteins, and small cells. Jet-freezing resembles plunge-freezing, but the cooling occurs by a jet of cold ethane directed to the sample. Jet freezing achieves a higher cooling rate, and can therefore be used for thicker samples. A CryoSol Vitrojet will be installed in the near future. For even thicker samples, like large mammalian cells or tissue, proper vitrification can be achieved by high-pressure freezing (HPF). Applying high pressure during freezing avoids ice crystallisation even when cooling rates are slower. After HPF, the samples can be used for further cryo-microscopy (for example cryoFIB-milling), or samples can be brought back to room-temperature by freeze-substitution. For rapid freezing, we utilise a Leica EM ICE high pressure freezer. For freeze substitution, we use a Leica EM AFS2 with an FSP automated reagent handling system.