Cellular energy ultimately derives form oxidation, with molecular oxygen being the predominate electron acceptor. This research project is directed toward the development of near infrared phosphorescence for determining oxygen concentrations in tissues. The specific goals are: 1. Phosphorescent probe molecules with optimal properties for oxygen measurements in biological systems will be designed. Probes that absorb light and phosphoresce in spectral regions where there is minimal absorption by biological pigments will be evaluated. Probes that remain in the circulatory system and are non-toxic to animals upon injection and/or are rapidly excreted to minimize the possibility of long term toxicity will be selected. Other probes will be designed to be taken up by cells and trapped internally, allowing direct measure of intracellular oxygen concentration. 2. The possible role of oxygen diffusion gradients in mitochondrial respiration will be examined. By comparing the oxygen dependencies of quenching of probes bound to respiring and non-respiring mitochondria, the effective oxygen depletion at the mitochondria can be determined. 3. Trans-plasma membrane oxygen differences will be examined in hepatocytes. To measure intra- and extra-cellular oxygen concentration two oxygen probe molecules will be used, one in the extracellular medium and the other trapped in the cellular cytoplasm. From differences in the oxygen dependence of the two, it will be possible to determine the barrier for oxygen diffusion imposed by the cell membrane and its associated unstirred layer. 4. Instrumentation will be developed for imaging the oxygen distribution in tissue. This will involve real time video imaging of phosphorescence intensity. Data processing will be used to obtain three dimensional images of the oxygen distribution in tissues. Eye lens, a transparent tissue where light scattering is not a problem, will be first examined. Later, rat kidney, a small but highly scattering tissue will be used.
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