Freeze-fracture methods: preparation of complementary replicas for evaluating intracellular ice damage in ultrarapidly cooled specimens.

Thin biological specimen sandwiched between conductive metal foils and ultrarapidly frozen in the absence of chemical pretreatments are well suited for ultrastructural studies by the freeze-fracture technique. However, the roughness of the metal surfaces, together with the thinness of the specimen, produce highly irregular surfaces upon fracturing, and thus yield fragile metal replicas. The techniques described here for stabilizing the replicas, initially with an open mesh grid and finally with a Formvar film, provide a means for obtaining a high percentage of intact replicas from which complementary images can be prepared. With the aid of the montages, complementary regions on the replicas can be located at some later time, usually in less than 1 hr. The complementary images obtained are excellent for evaluating the factors which influence the quality and resolution of the replicas--such as heating and plastic deformation--and for describing the preservation of biological structures. Regions within the specimen which display unacceptable ice crystal growth or damage can be easily and confidently identified in complementary images.