Platinum shadowing, complementary imaging and stereo analysis: Techniques developed for TEM can now be used to increase the resolution of frozen hydrated specimens in a SEM

Abstract

In 1957, Steere froze virus crystals, fractured and transferred them to a vacuum evaporator where they were etched, shadowed and coated. The coating, which was then removed from the tissue and mounted on a grid, represented a replica that could be observed in the transmission electron microscope (TEM). This accomplishment is regarded as the first successful biological application of the freeze-etching technique in electron microscopy. This technique was further advanced by describing procedures whereby the replicas from both surfaces of a frozen, fractured sample could be recovered. This pair of replicas provided additional surface information by allowing one to view and compare the two opposing surfaces, or the complementary images, of a freeze-fractured biological specimen. Using a goniometer stage the replicas were also photographed, tilted and rephotographed to produce stereo pairs that could be used to analyze three-dimensionally the surface characteristics of the replica. Until recently these TEM techniques have been the principle procedures utilized by biologists to gain high resolution structural details about virus crystals, biological membranes and a wide range of other material.