Simple cyropreservation techniques to preserve cellular ultrastructure of freeze fracture or sectioning prior to image analysis

Summary form only given. Computer-assisted computer imaging techniques are limited in their value by the extent to which the structure being analyzed deviates from its native state. For intracellular structures, traditional chemical fixations have been well established to leave substantial room for deviation from reality. For example, membrane-bound vesicles can be relocated or abnormally fused and molecules can be extracted. In addition, traditional techniques can substantially diminish antigenicity in immunocytochemical localization protocols. Although the alternative of freeze-substitution and ultra-low temperature embedding are established in the literature, many people have not switched to these techniques. For structures deep within a tissue (up to about 25 μm), these techniques will require an expensive high-pressure freezer (approximately $150,000.000). However, for structures on the surface of a tissue block, small organisms, or single cells in suspension, we describe fairly simple apparatus and reliable techniques to achieve ultra-rapid freezing routinely ( up to about 30 μm). In addition, we describe preliminary efforts to use computer-assisted electron tomography to reconstruct three dimensional structures from freeze fracture and sectioned images