Cryofixation and cryosubstitution: a useful alternative in the analyses of cellular fine structure

A study of the fine structure of cultured mouse P815 cells as well as of mouse liver tissue after having undergone cryofixation and cryosubstitution is reported here. The P815 cells were cryofixed by a projection onto a liquid nitrogen (LN2), or liquid helium (LHe), cooled copper mirror: the liver tissue was processed (cryofixed) by high pressure freezing using a Balzers HPM 010 apparatus. No conventional chemical fixatives were used in the substitution medium which consisted of pure acetone. Embedding was carried out either in Lowicryl K11M, Lowicryl HM23, Epon or in LR White resins. The results of this study enabled us to conclude that a) one can obtain good preservation of cellular ultrastructure using different cryofixation methods and cryosubstitution without the use of chemical fixatives: b) different methods of embedding can be applied after various cryofixation techniques giving rise to slight differences in the well preserved fine structure; and c) high pressure freezing is to be recommended, in cryosubstitution studies, over that of slam freezing especially when cryofixing larger pieces of tissue which can result in a good morphology of up to 400 microns in depth.