VITRIFICATION DEPTH CAN BE INCREASED MORE THAN 10-FOLD BY HIGH-PRESSURE FREEZING

Biological specimens prepared for cryoelectron microscopy seem to suffer less damage when they are frozen under 2 kbar pressure rather than under normal conditions. The volume that can be well preserved is larger. This fact has been illustrated in a number of publications on a number of different samples. However, there is a lack of quantitative data concerning the depth of this good specimen preservation. Catalase crystals in various sugar solutions have been used as test objects and vitrification, as determined by electron diffraction, has been used as the criterion for good freezing. Keeping all other conditions equal, the depth of vitrification is approximately 10 times larger with freezing at high, rather than normal, pressure. The high-pressure vitrification depth in a 15-20% sugar solution averages 200 mu m. Fully vitrified specimens up to 700 mu m in thickness are obtained. When crystalline water is observed it is frequently in the form of high-density ice rr, III or IX. These results are probably also relevant for typical biological specimens. The advantage of high-pressure freezing must be balanced by the possible consequences of a considerably increased cooling time and by the damage that may be induced by the pressure.