A new technique for removal of amorphous phase tissue water without ice crystal damage: a preparative method for ultrastructural analysis and immunoelectron microscopy.

An apparatus has been produced that can remove amorphous phase tissue water via molecular distillation without devitrification or rehydration. This method represents a fundamental advance in tissue preparation, making possible for the first time ultrastructural localization of soluble molecular entities without the problems of alteration, re-distribution, and loss which have plagued conventional techniques. Fresh slices of rat brain, liver, or kidney, and monkey retinal tissue were cryofixed by bounce-free, metal mirror cooling on copper bars immersed in liquid nitrogen (LN2). Tissue transferred under LN2 was then placed in a precooled copper specimen block, which was subsequently lowered into a LN2-cooled stainless steel chamber. After rough pumping at 1 X 10(-3) mbar with a mechanical pump to remove LN2, the chamber was evacuated with a cryopump or turbomolecular pump to achieve a hydrocarbon-free, ultra-high vacuum of 1 X 10(-8) mbar. Equilibrium temperature in the chamber before the drying cycle was -192 degrees C. The copper specimen block was equipped with a thermocouple and a programmable feedback-controlled heating circuit. Tissue was dried by increasing the specimen block temperature 1 degree C/hr during the critical drying phase while monitoring the rate of water removal with a partial pressure analyzer. Results obtained indicate that drying is complete below the devitrification temperature of amorphous phase tissue water. Dried tissue was fixed with osmium tetroxide vapor, vacuum-embedded in a low-viscosity epoxy resin, sectioned, stained, and viewed with the electron microscope. Processed tissue exhibits excellent morphological preservation without the use of pre-fixation or cryoprotective agents. Thin sections of this tissue are excellent for immunocytochemical staining and electron microprobe analysis.