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 185K in organic solvent. Temperature elevation of cryosubstituted E. coli cells, when still in organic solvent, had no effect on the preservation of chromatin structure. The achieved stabilization was found to be established independent of the presence of the chemical fixative. Cryosubstitution without the use of a chemical fixative allows for excellent preservation of cellular ultrastructure. Beyond that, the approach is preferential for sensitive antigens in immuno-electron microscopy. We conclude that low-temperature dehydration by cryosubstitution in organic solvents is able to form stable cross-links between macromolecules by hydrophobic interactions.
Bohrmann
B
B
Preclinical Research, F.Hoffmann-La Roche Ltd., CH-4070, Basel, Switzerland. bernd.bohrmann@roche.com
Kellenberger
E
E
D015925:Cryopreservation / Q000379:methods*; D004926:Escherichia coli / Q000648:ultrastructure*; D007150:Immunohistochemistry; D008854:Microscopy, Electron / Q000379:methods*; D016707:Tissue Fixation / Q000379:methods