Cryosubstitution of frozen biological specimens in electron microscopy: use and application as an alternative to chemical fixation

Frozen bacterial cells were low-temperature embedded after cryosubstitution at 185 K in organic solvent. Temperature elevation of cryosubstituted E. c oli cells, when still in organic solvent, had no effect on the preservation of chromatin structure. The achieved stabilization was found to be establi shed independent of the presence of the chemical fixative. Cryosubstitution without the use of a chemical fixative allows for excellent preservation o f cellular ultrastructure. Beyond that, the approach is preferential for se nsitive antigens in immunoelectron microscopy. We conclude that low-tempera ture dehydration by cryosubstitution in organic solvents is able to form st able cross-links between macromolecules by hydrophobic interactions. (C) 20 00 Elsevier Science Ltd. All rights reserved.