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 ade quate cryoimmobilization of biological samples thick enough to describe the bulk of the sample. In the current state of HPF instrumentation and prepar ation 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 re quired. (ii) There is mechanical difficulty in cutting such thin samples wi thout 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 alteration s. 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 nat discoid samples s andwiched between relatively thick aluminium platelets. Based on this fact, we developed a prototype of a new microbiopsy device which enables the qui ck excision of such cylinders of soft tissues. The device utilizes sharp go ld needles of an inner diameter of 200 mu m and wall thickness of 50 mu m. The frozen sample contained in the soft gold needle permits all the manipul ations needed for conventional cryo-preparation techniques for electron mic roscopy (e.g. cryo-sectioning, freeze-fracturing, freeze-substitution).