Cryoultramicrotomy as a preparation method for x-ray microanalysis.

X-ray microanalysis allows "in situ" studies on ;the elemental composition of the cell and its compartments. The reliability of the results is almost entirely determined by the preparation method. Two criteria for a reliable preparation method have been formulated: (1) it must preserve the ultrastructure to permit identification of cellular compartments, (2) it must retain the elements in their in vivo location. We investigated cryoultramicrotomy and confirmed its validity as a preparation method for X-ray microanalysis of diffusible elements. The method is only truly reliable, however, under the following conditions: (1) the tissue must be frozen rapidly, (2) the sections must be cut in the ice crystal free surface layer, (3) the sections must be cut at low temperature (e.g. a specimen temperature of -140 degrees C, a knife temperature of -100 to -120 degrees C and a cryochamber temperature under -100 degrees C, and pressed onto the grid and freeze-dried at a temperature of -100 degrees C).