Preparation of biological tissues for electron microscopy by freeze-drying

A dependable method for freeze-drying tissues for electron microscopy has been developed. Thin slices of fresh tissue were frozen by bringing them into direct contact with a polished copper bar at liquid nitrogen temperature. The tissue was transferred to a copper specimen block equipped with a thermocouple and heating circuit for accurate control of the environmental temperature of the tissue, and evacuated in a glass freeze-drier using clean high vacuum techniques for keeping the system free of hydrocarbons. The tissue was dried by increasing the temperature of the specimen block 10 degrees C each hour while monitoring the rate of water removal from the tissue with a partial pressure analyzer. The dry tissue was fixed with OsO4 vapor, vacuum embedded in a low viscosity epoxy resin, sectioned, stained, and viewed with the electron microscope. Tissue processed in this manner exhibits excellent morphological preservation at both cellular and organellar levels without prefixation or the use of cryoprotective agents. The results of the experiments using the partial pressure analyzer indicate that small blocks of tissue can be dried in a short time at low temperature.