Immersion freezing of suspended particles and cells for cryo-electron microscopy.

Immersion freezing of thin aqueous specimens is an essential preparation technique for cryo-transmission electron microscopy (cryo-TEM), aiming to preserve fragile biological structures such as molecules and cells in their hydrated environment for a close-to-native visualization. For successful experiments, vitreous ice must be produced, surface contamination must be avoided, and, most important, the natural state of the structure must be preserved. This protocol describes immersion freezing of biological samples, such as purified protein complexes, viruses, liposomes, synthetic cytoskeletal filaments, isolated organelles, or small cells suspended in an aqueous solution, using the new Leica “EMGP” grid plunger. It includes a discussion of issues of general importance for cryo-EM, such as the properties of the sample and the pretreatment of the specimen carrier. It also provides details on how to make the most of the special features of this instrument to obtain good specimens and reproducible results. Troubleshooting issues concerning the operation of the GP in particular, as well as common problems in immersion freezing encountered on manual and semiautomatic instruments, are addressed.