On optimizing high-pressure freezing: from heat transfer theory to a new microbiopsy device.

High-pressure freezing (HPF) is currently the only method which enables adequate cryoimmobilization of biological samples thick enough to describe the bulk of the sample. In the current state of HPF instrumentation and preparation methods, the technique has not yet reached its full potential. While suspensions can be prepared easily for HPF, tissue preparation is restricted by the need to compromise between different requirements and difficulties. (i) In order to achieve optimal freezing quality, very thin samples are required. (ii) There is mechanical difficulty in cutting such thin samples without distorting the organization of the tissue. (iii) The cutting and the succeeding preparation steps of small samples require long handling times (minutes), which may result in physiological and hence structural alterations. Computerized heat transfer simulations are presented which confirm that the efficiency of heat extraction from cylindrical samples contained within thin-walled metal tubes is higher than from standard flat discoid samples sandwiched between relatively thick aluminium platelets. Based on this fact, we developed a prototype of a new microbiopsy device which enables the quick excision of such cylinders of soft tissues. The device utilizes sharp gold needles of an inner diameter of 200 microm and wall thickness of 50 microm. The frozen sample contained in the soft gold needle permits all the manipulations needed for conventional cryo-preparation techniques for electron microscopy (e.g. cryo-sectioning, freeze-fracturing, freeze-substitution).