An apparatus for substitution of water in frozen tissue at regulable low temperature

Summary

A simple apparatus for freeze substitution at regulable temperatures down to — 130°C is described.

In principle, the apparatus consists of a can containing the tubes with the substitution fluid. The can is placed in a cylinder of insulating material, the lower part being surrounded by the cooling medium, i.c. liquid air. The desired temperature in the can is thermostatically controlled by a heating element. Calculations are given for the relation of the heat input and output to the cooling medium and the conductive properties of the materials used. On this basis, the construction conditions (thickness of insulation; level of the liquid air; etc.) are chosen such that:

• 1. the consumption of liquid air is low;

• 2. the heat transport from inside the can to the liquid air is sufficient to maintain the inner temperature at the desired level.

With this construction, the consumption of liquid air (which is compensated for automatically) at an inner temperature of the can of −130°C or −70°C amounts to four and six litres per day respectively.

The problems, still unsolved with respect to the application of freeze-substitution specially in electron microscopy are considered from the theoretical and practical point of view. The authors' electron-microscopical observations on the morphology of fresh frozen sections (50 μ) fixed in OsO₄ are mentioned. These observations indicate that the circumstances of substitution are more critical for morphological preservation than are the circumstances of freezing, and that for electron microscopy frozen sections can probably be used instead of frozen tissue blocks to reduce the time of substitution. It is pointed out that the use of the apparatus described here may help in solving these problems.