Pretreatment of samples and freezing

1. Instrumentation

Equipment (an instrument for freezing, an instrument for freeze-substitution), available from various companies, is needed which allows rapid freezing of specimens followed by substitution of the ice in the sample by an organic solvent. This has to be done at temperatures which avoid damage by recrystallization of the frozen water in the sample. An automatically controlled apparatus for freeze-substitution is a major advantage.

2. Sample pre-treatment

Depending on the type of specimen (isolated cell components, cells, tissues), addition of fixatives such as glutaraldehyde (up to 30%, v/v) uranyl acetate (0.5-2%, w/v) or osmium tetroxide (0.5-1%, w/v), (Slot et al., 1988) and of glycerol for cryoprotection (Robards and Sleytr, 1985; Müller, 1988) may be necessary. These additions should be applied at concentrations which, together with the buffer system, are in sum isotonic to the osmotic state of the sample. Optimization is necessary, especially when plant material is to be examined. A considerable amount of data in this respect has accumulated, derived in particular from the field of freeze-etching and freeze-fracturing (Müller, 1988).

3. Freezing

A drop of liquid containing the pre-treated sample is put on a copper grid; its volume should be as low as possible (around 1 mm3); note that the most rapid freezing only takes place close to the surface of the specimen; inside, the cooling rate is considerably lower. Now the sample is quickly dipped into propane (which again is cooled by liquid nitrogen) by use of pre-cooled tweezers, removed, and immediately transferred to liquid nitrogen to avoid warming up (Dubochet, 1984).

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