Low temperature techniques applied for CTEM and STEM analysis of cellular components at a molecular level

One of the most important problems in tissue preparation for electron microscopic analysis at a molecular level involves the preservation of the tissue without introducing extensive denaturation of the proteins. Low temperature is a most efficient condition for the inhibition of protein denaturation and freeze-drying offers favourable conditions for transferring proteins to a dry state with minimal denaturation of the proteins. However, the embedding of the dried tissue in a plastic leads to extensive denaturation of the proteins when performed in the conventional way. This eliminates very efficiently the advantages of the method. The situation becomes even worse when subjecting the tissue to freeze-substitution. To eliminate as far as possible the denaturing effect of plastic embedding, freeze-drying can be combined with low temperature embedding in a plastic. Freeze-fracturing allows a most efficient use of low temperature to reduce conformation changes in proteins. The value of the freeze-fracturing technique depends entirely on a precise knowledge of the location of the fracture planes. Since this location is not known, it must be determined on the basis of a deduction. If this deduction is wrong, the method becomes misleading. Two methods which allow a certain testing of the correctness of the deduced location of the fracture planes are mentioned.