The Distribution of Light Elements in Biological Cells Measured by Electron Probe X-Ray Microanalysis of Cryosections

Abstract

The intracellular distribution of the elements carbon, nitrogen, and oxygen was measured in cultured rat hepatocytes by energy dispersive electron probe X-ray microanalysis of 100-nm-thick freeze-dried cryosections. Electron irradiation with a dose up to 106e/nm2caused no or merely negligible mass loss in mitochondria and in cytoplasm. Cell nuclei lost carbon, nitrogen, and—to a clearly higher extent—oxygen with increasing electron irradiation. Therefore, electron doses less than 3 × 105e/nm2were used to measure the subcellular compartmentation of carbon, nitrogen, and oxygen in cytoplasm, mitochondria, and nuclei of the cells. The subcellular distribution of carbon, nitrogen, and oxygen reflects the intracellular compartmentation of various biomolecules. Cells exposed to inorganic mercury before cryofixation showed an increase of oxygen in nuclei and cytoplasm. Concomitantly the phosphorus/nitrogen ratio decreased in mitochondria. The data suggest mercury-induced production of ribonucleic acid (RNA) and decrease of adenosine triphosphate (ATP). Although biomolecules cannot be identified by X-ray microanalysis, measurements of the whole element spectrum including the light elements carbon, nitrogen, and oxygen can be useful to study specific biomolecular activity in cellular compartments depending on the functional state of the cell.