The design and use of a simple device for rapid quench-freezing of biological samples

The detailed design of a simple device for rapid quench-freezing of biological samples under reproducible conditions is presented. With spring-augmented descent, sample immersion velocity of 10 m s-1 into a cryogenic liquid is achieved. Biological samples, loaded in Balzers planchets, Denton holders, or a newly designed 'titanium envelope', are suitable for rapid-freezing with this device. Using 4 micrometers titanium foil, light weight (1 mg) streamlined holders can easily be made to enclose cell suspensions or tissue samples. The foil envelope is designed for efficient heat dissipation while protecting the sample from possible impact or flow distortions occurring from spring-augmented immersion. Human erythrocytes, quench-frozen in the titanium envelope, were prepared for electron microscopy by the freeze-substitution technique. Two opposing 25--30 micrometers surface zones were frozen in the apparent absence of ice. The extended depth of cryofixation is attributed to the advantages of thin foil in the titanium envelope design and the use of rapid-immersion technique.