Samples are either rapidly frozen generating a frozen hydrated sample for imaging or further processing or may be chemically fixed and cryo-protected before freezing to avoid damage caused by ice crystal formation. In addition to avoiding potential artefacts associated with some other forms of sample preparation, cryo-preserved samples exhibit the best antigenicity, essential for successful immuno-EM.
Examples of cryo-techniques include:
1. Cryo-ultramicrotomy
Ultra thin (50-70 nm) frozen sections, usually of fixed, cryo-preserved samples are prepared for subsequent immuno-labelling with gold conjugated antibodies. Two Leica cryoultramicrotomes are available in the lab.
2. Cryo-substitution
A cryo-variant of conventional resin embedding that has the advantage of enhanced antigenicity for immuno-labelling with a more conventional view of ultrastructure associated with resin-embedded samples. Samples are frozen and dehydration and resin embedding are performed in a cryo-substitution unit typically at -90°C in acetone. The reduced protein denaturation produced by sublimation of water with acetone at these low temperatures preserves antigenicity beyond that normally associated with resin-embedded samples. Low viscosity resins (eg. Lowicryl) that can be polymerised at low temperatures with UV light are used to encase the sample for subsequent sectioning and immuno-labelling. 2 Leica automated freeze substitution apparatus are available in the lab.
3. Frozen hydrated TEM:
Unfixed tissue fragments or sample suspensions are rapidly frozen to form under conditions promoting vitreous ice rather than crystalline ice formation to prevent damage to the sample. This technique is often used to image liposome suspensions, multi-molecular complexes and viruses for 3-D reconstruction. Samples prepared in this way are usually extremely sensitive to electron beam damage, and the area of interest is often destroyed in the process of capturing the image. A variety of approaches have been developed to improve the chances of generating sharp images of these samples. A Vitrobot and a Leica EMPACT2 high pressure freezer are available.
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