Freeze-substitution of chemically stabilized samples for biological field emission scanning electron microscopy

The high resolution imaging capabilities of modern field emission scanning electron microscopes require adequately improved tissue preparation procedures to prevent the collapse of macromolecular structures and the extraction of molecules. A routine cryo-stabilization technique is described which utilizes chemical crosslinking and cryo-dehydration for mechanical and chemical stabilization of protein and lipid structures and increase of electrical conductivity of the sample. Thiocarbohydrazide (TCH) serves as a general mordant for osmium tetroxide crosslinking. However, extensive washing after all impregnation steps is necessary to dissolve unspecific osmium black precipitations at the sample surface. Collagen I aggregates showed increased stability against collapse after TCH osmification alone, whereas pulmonary surfactant liposomes require additional freeze-substitution in methanol and Freon 113 for stabilization during critical point drying. Environmental scanning electron microscopy (at water vapor pressures of 5-10 torr within the specimen chamber) was used to control, in the wet phase, the stabilization procedure at the level of chemical crosslinkage. It could be confirmed that tannic acid, often used to stabilize lipids, leads to artificial rearrangement of bilayered liposomes into compact presumable multilayered bodies, whereas the TCH osmification preserved liposome structures and their aggregates. The increase of electrical conductivity of sliced tissue was demonstrated on kidney. Support technologies for the cryo-stabilization procedures are described in detail, as well as simple routines for first stabilization trials with new samples. On pulmonary tissue, the excellent preservation of alveolar shape and fine structures of intermediate forms of surfactant are described.