Ultra-rapid freezing of thin biological samples

Miniature thermocouples are used to follow the time course of cooling within test samples plunged into liquid coolants. Variations in the temperature and type of coolant (propane, Freon 22, sub-cooled liquid nitrogen), in the velocity and depth of plunge, and in the size and shape of samples were investigated. Coolants have their optimum cooling properties at their lowest liquid temperatures but show different dependencies on temperature. The optimum velocity of plunge depends on the shape of the sample. The strongest influence on cooling rate comes from the size and shape of the sample. Spherical samples about 1 mm3 show rates < 500 decrees C/s while samples 0.001 mm3 show rates > 50,000 degrees C/s for freezing in liquid propane under identical conditions. Thin samples of equivalent volume can be more rapidly frozen. Cooling rates of about 11,900 degrees C/s (0 degrees C to -100 degrees C) are recorded for thin samples (0.1 mm3 and 20 micrometers thick) sandwiched between copper strips and rapidly plunged into liquid propane (-190 degrees C). An apparatus is described for using these holders in freeze-fracture studies. Thin samples thus frozen show no evidence of ice crystal growth in either low-temperature x-ray diffraction patterns or in freeze-fracture electron micrographs.