Cryotechniques are a group of related procedures for stabilizing, or “fixing” specimens for microscopic observation. Samples are rapidly frozen to maintain cellular structure and composition as it exists under physiological conditions, such as electrolyte concentration and protein antigenicity. Alternatively, samples may be lightly fixed and cryoprotected first before freezing. These techniques preserve the native structures of tissues without the artifacts associated with chemical fixation. Some cryotechniques facilitate investigations of membranes and membrane proteins.
Examples of cryotechniques include:
1. Cryo-ultramicrotomy—Ultra thin frozen sections cut for the TEM. Generally regarded as the most sensitive TEM immuno prep technique.
2. Freeze Fracture—Samples are fixed, frozen, fractured and a replica of the fracture surface is imaged in the TEM. Usually performed on cells or lipid suspensions. It is one of the only ways to demonstrate the presence of tight junctions.
3. Frozen hydrated TEM—A thin layer of unfixed aqueous sample is rapidly frozen to form a sheet of vitreous ice with the sample suspended in it. This technique is often used to image liposome suspensions and record images of viruses for 3-D reconstruction.
4. Cryosubstitution—This technique avoids the denaturing effects of room temperature dehydration and resin infiltration. Samples are frozen and transferred to the Cryosubstitution unit and placed in, typically, acetone at -90°C. At this low temperature the water ice is replaced by the acetone in a sublimation type process thereby minimizing adverse effects on antigenicity. The sample is then infiltrated with special low viscosity resins (such as the Lowicryl series) and cured by UV light.