Freeze-drying of Biological Specimens for Electron Microscopy Using Thermoelectric Cooling

Freeze-drying for electron microscopy is one of the most successful methods for avoiding specimen distortion caused by drying of liquid solutions in air. It has been calculated that the surface forces of receding droplets subject small specimens to pressures of the order of tons per square inch (1). In freeze-drying, an aqueous solution of the specimen is rapidly frozen and the ice allowed to sublime under vacuum. Thus, the specimen is maintained in a rigid state and the surface tension forces are greatly reduced. Several studies have demonstrated a well preserved three-dimensional structure in frozen dried material, as compared to the same material dried in air (2-5). However, freeze-drying has not been widely used, largely because of inherent difficulties associated with existing methods which necessitate the use of separate vacuum systems, special glassware, and glass-to-metal vacuum seals. Relatively large areas of the apparatuses become cold and act as water vapor traps for the atmosphere so that several hours are often required for complete sublimation. Most methods and equipment used to date involve transfer of the frozen dried specimen from the freeze-drying apparatus through the atmosphere to a vacuum evaporator for replication and/or heavy metal shadowing. Even if the specimen is warmed before transfer, absorption of water and consequent distortion of the specimen may