Premium
Freeze‐fracturing at low temperatures: I. A device for fracturing biological specimens at 77–10 K under high vacuum
Author(s) -
Niedermeyer W.
Publication year - 1982
Publication title -
journal of microscopy
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.569
H-Index - 111
eISSN - 1365-2818
pISSN - 0022-2720
DOI - 10.1111/j.1365-2818.1982.tb00347.x
Subject(s) - liquid nitrogen , ultra high vacuum , outgassing , materials science , contamination , composite material , chemistry , nanotechnology , ecology , organic chemistry , biology
SUMMARY Standard freeze‐etching or freeze‐cleaving is performed at 173 K in a vacuum of 133 μPa or at 77 K under liquid nitrogen with subsequent transfer of the specimen into a vacuum chamber. It has been suggested that the frequent poor resolution of morphological details, the poor complementarity of innermembrane protein particles and the semi‐crystalline substructures in biomembranes are caused by structural distortion or plastic deformation due to sheer forces which occur even at 77 K during fracturing or cleaving. In addition, water contamination and radiant heat damage occurring during replication introduce artefacts to the structural record. These artefacts could be avoided or reduced by lowering the temperature at which fracturing or cleaving and shadowing is carried out, to about 10 K. Therefore, a device for cleaving biological specimens at 15–10 K under high vacuum was constructed. To allow the use of existing equipment, the device was built into a standard Balzers 301 vacuum unit, where the specimen transfer is done via an airlock system which allows hoar frost contamination free transport of the specimen holder onto the specimen table. To reduce or prevent the condensation of water and other residual gases in the vacuum onto the freshly cleaved specimen surface at 10 K, the specimen is surrounded by two cooled surfaces of 6 and 20 K. All condensable gases outside those shielding shrouds will condense on these surfaces before reaching the specimen. This makes it possible to work at a high vacuum of 3 μPa outside the cooled shrouds, which can be reached with standard turbomolecular pumps. The actual vacuum within the cooled shrouds is estimated to be approximately 13 nPa. Residual gas analysis before and during replication reveals equal conditions to ultra high vacuum systems. An analysis of the yeast cell paracrystalline plasmalemma structure shows that the topographic resolution of the crystalline arrays has been improved by working at 12 K. However, plastic deformation still occurs under these conditions. This observation points to the possibility that what is described as plastic deformation, for at least some membrane proteins, may be a loss of resilience at low temperatures.