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Complementary freeze‐fracture, freeze‐etch specimens
Author(s) -
Steere Russell L.,
Erbe Eric F.
Publication year - 1979
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.1365-2818.1979.tb01177.x
Subject(s) - cryoprotectant , freeze drying , liquid nitrogen , vesicle , ice crystals , materials science , fracture (geology) , sucrose , etching (microfabrication) , dislocation , crystallography , membrane , biophysics , chemistry , composite material , chromatography , biology , cryopreservation , optics , biochemistry , organic chemistry , embryo , physics , layer (electronics) , microbiology and biotechnology
SUMMARY A procedure is described for the preparation and comparison of complementary freeze‐fracture‐freeze‐etch specimens. These complementary replicas reveal the value of etching some specimens even when the cryoprotectant concentration amounts to as much as 25% glycerol and 25% sucrose. As expected, the peplomers on the outer surface of the surrounding membrane of potato yellow dwarf virus are clearly revealed on the etched specimens but nearly invisible in the complementary freeze‐fracture specimen. Unexpected differences in the internal structure of vesicles within a freeze‐etched chloroplast have been revealed. The cross‐fractured surfaces of some other vesicles have a rough structure, whereas the complementary freeze‐etch surfaces, also shadowed at 123 K, are extremely smooth, suggesting that the contents must have been liquid at 175 K. Large ice crystals form within tobacco mosaic virus (TMV) crystals in infected cells frozen rapidly without cryoprotectant. Cryoprotectant consisting of 25% glycerol and 25% sucrose prevents formation of ice crystals when these specimens are frozen under the same conditions. When cryoprotectant concentrations are decreased from the above level, ice crystals within the TMV crystals are often clearly demonstrated in the freeze‐etch specimen but not in the complementary freeze‐fracture specimen. These results suggest that the complementary freeze‐fracture‐freeze‐etch procedure and the extremely fragile TMV crystalline inclusions are ideal for examining the value of various cryoprotectants and the effect of freezing rates, etching temperatures, and etching times on the final image.