Most biological samples contain 70-95% water, consequently cryofixation and freeze-fracturing result in relatively smooth surfaces that exhibit few structural details. Freeze-etching, a technique that solved this problem, was initially developed for TEM observations of virus particles by Steere nearly 40 years ago. The technique, which sublimes water-ice from the surface of a fractured sample, produces surface topography that corresponds to the structural components on the freeze-etched face. This technique was further enhanced by recovering the complementary halves of a fractured sample, etching one of the surfaces and then comparing the complementary replicas from the freeze-fractured and freeze-etched faces. Recently, similar techniques were used on frozen, hydrated samples to examine complementary halves of freeze-fractured, freeze-etched specimens by low temperature SEM. Imaging complementary images of frozen, hydrated specimens in the SEM was faster than imaging complementary replicas in the TEM, however the procedure required specialized holders and was technically demanding.To simplify comparisons of freeze-fracture, freeze-etch images, samples were frozen, fractured and etched in the prechamber of an Oxford CT 1500 HF Cryotrans system that was attached to a Hitachi S-4100 FESEM.
Navigating the Fog: A Correlated XRM and FIB-SEM Imaging Pipeline for the Rapid and Precise Spatial Targeting of Rare Structures in Biological Samples
