Comparative study of freeze-substitution techniques for X-ray microanalysis of biological tissue

A comparative study of some aspects of freeze-substitution as a preparative method for X-ray microanalysis of diffusible ions in biological specimens was carried out. Four substitution fluids were compared with each other and with cryosections. As criteria, elemental ratios as determined by X-ray microanalysis of thin sections of mouse pancreas were used. Freeze-substitution for 3 weeks in diethyl ether or for 2 days in tetrahydrofurane gave results comparable to those obtained in cryosections. Slow warming to room temperature after freeze-substitution in tetrahydrofurane gave slightly better results than fast warming. Freeze-substitution in acetone resulted in noticeable loss and redistribution of ions and freeze-substitution in methanol gave very poor results.