Studies on cellular structure and ice location in frozen organs and tissues: the use of freeze-substitution and related techniques.

Recent studies have led to the conclusion that extracellular ice per se can damage whole organs and tissues. Thus information on the amount and distribution of ice is an important factor in the design of cooling regimens that avoid intracellular ice formation and attempt to localize the ice formed in areas of the tissue where its disruptive effects can be minimized. Furthermore, ultrastructural studies at subfreezing temperatures can enhance the interpretation of information gained from morphological and function studies conducted before cooling and after rewarming. Although many techniques exist for observing and recording structure in the frozen state, not all are applicable to tissues or organs. Freeze-substitution and isothermal freeze-fixation provide two flexible techniques to explore the frozen state. Isothermal freeze-fixation is most suitable for studies close to the melting point, while freeze-substitution can be used at lower temperatures, extending as far as -120 degrees C. A careful choice of technique can provide an accurate assessment of the amount and distribution of the ice phase and the structure of the tissue matrix.