Scanning Electron Microscopy


An apparatus able to remove amorphous phase tissue water without recrystallization or rehydration has been produced. Application of this technique to biological samples achieves both the preservation of ultrastructure and the retention of cellular macromolecules and solute without redistribution or modification.

Small pieces of fresh tissue were cryofixed by the method of bounce free metal-mirror freezing on polished copper bars at liquid nitrogen temperature. Tissue samples were then placed under liquid nitrogen in a copper sample holder equipped with a thermocouple and feedback controlled heating circuit. Under liquid nitrogen the sample block was placed in a stainless steel sample chamber which was then evacuated to a hydrocarbon-free ultrahigh vacuum (1x10-8mbar). Equilibrium temperature prior to the onset of the drying cycle was -192°C. Tissue was dried by increasing the temperature of the specimen block 1.33°C each hour while monitoring the rate of water removal with a partial pressure analyzer. Results indicate that drying is complete below the devitrification temperature of amorphous phase tissue water. After drying, tissue was fixed with osmium tetroxide vapour, vacuum embedded in low viscosity epoxy resin, sectioned, stained and viewed with the electron microscope. Tissue processed in this manner exhibits excellent morphological preservation with out the need for prefixation or cryoprotective agents. In addition, by avoiding prefixation and solvent contact during resin embedding, this method provides the basis for combining ultrastructural preservation with optimum material for immunocytochemical staining and elecron microprobe analysis.

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