X-ray microanalysis of calcium in ischemic myocardium: a new method of rapid freezing.

X-ray microanalysis of biological material is best accomplished on unfixed frozen tissue sectioned on a cryoultramicrotome. The need for ultra-rapid freezing to minimize ice crystal artifact has been well documented but is difficult to achieve without the use of potentially hazardous Isopentane or Freon 22 in liquid nitrogen slush. In the present study we employ a slurry of powdered graphite in liquid nitrogen to obtain rapid freezing of cardiac tissue. The results obtained were as good as those which are achieved with Freon 22 in liquid nitrogen, and subsequent cryoultramicrotomy produced thin sections relatively free of ice crystal artifact. Microanalysis revealed a slight increase in mitochondrial calcium as compared with cytoplasmic calcium in normal myocytes. Mitochondrial calcium concentration rose significantly after 30 min of ischemia while only a slight rise in cytoplasmic calcium occurred. These findings suggest that the techniques employed are adequate for detecting ion shifts which occur during ischemia and may be useful for determining the effects of therapeutic agents at the organellar level.