Improved cryofixation applicable to freeze etching.

Freeze etching of solute model systems (e.g., glycerol or ferritin solutions) demonstrates that cryofixation can introduce serious artifacts due to the segregation of the dissolved or dispersed material from the solvent. Since, in principle, this problem can be reduced by increasing the cooling rate, a new technique has been developed which combines spray freezing with freeze etching. This spray-freeze-etching is applied by first spraying the specimen into a liquid cryomedium. The frozen droplets are then "glued" together with butylbenzene to form a regular freeze-etch specimen, while the temperature of the sample is kept at -85 degrees C. The results obtained by spray-freeze-etching are far superior to those obtained by standard freezing. Our results, using 5% glycerol as a test specimen, are equivalent to those obtained by the high-pressure method (1). The reduction of segregation during freezing makes freeze etching a method applicable for the investigation of solute systems. Furthermore, the study of unicellular organisms or cellular fractions by freeze etching without the use of antifreeze is made possible.