Sample vitrification

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.