Simple techniques for freeze clamping and for cutting and milling of frozen tissue at low temperature for the purpose of two- or three-dimensional metabolic studies in vivo.

Abstract A technique is described for freeze-clamping of parenchymal tissue (e.g., liver) which causes the tissue to be rigidly fixed to an aluminium cup in the frozen state with a well-defined, reproducible orientation of the tissue as well as a minimum of morphological distortion of the major part of the sample. Furthermore, three instruments for low-temperature cutting or milling of the frozen sample for the purpose of two- or three-dimensional metabolic studies are described. The cutting and milling instruments work according to the principle of ordinary workshop machines for steel work. The frozen sample fixed in the aluminium cup may be mounted in the milling instrument and cut at the temperature of liquid nitrogen with high precision; e.g., one instrument may be adjusted to mill off tissue layers of a thickness of only 20 μm. Thermocouple readings from the frozen sample suggest that the milling process does not cause significant heating of the sample. This is further supported by the fact that the amount of labile metabolites, ATP, ADP, AMP, lactate, and pyruvate, is unaffected by the milling process.