A New Concept and Machine for High Pressure Freezing

Abstract To date, high pressure freezing is the only reliable cryofixation method for vitrifying bulk biological samples up to 150μm in thickness (1). This proves the enormous potential of the method. Nevertheless, the procedure is not well accepted by the scientific community. It seems that people not completely convinced by the method have reservations to use high pressure freezing machines. The main reasons seem to be that the commercially available machines are noisy, large, very heavy, expensive and consume a lot of liquid nitrogen. Last but not least, a relatively large volume of cryogen is pressurized to 2000bar representing a potential risk of accident. Considering high pressure freezing to be a very potent method, it was tried to establish a new machine avoiding the above mentioned disadvantages. The first functional high pressure freezing apparatus was freezing fine metal rods with a boring of 0.2mm in diameter (2). This boring was filled with suspensions, the rod was connected to a pressure generator thus pressurizing the suspension, and simultaneously the metal rod was frozen from the outside by a low pressure jet of liquid nitrogen. The big disadvantage of the method was that only freeze-fracture experiments could be performed at the time because the sample could not be removed from the boring. As a consequence, this approach was never commercialised. In the mean time sample preparation has improved. Cellulose capillaries can be used to freeze suspensions and tissue samples (3,4). To freeze these capillaries in the new machine, they are introduced in a sample holder which consists of a copper tube with an inner diameter of 0.3mm and a length of 16mm. This tube fits into a concentrical stainless steel tube which stabilises the copper rod and helps to fix the sample in the cryofixation device.