Fusinite as a specific probe for the determination of molecular oxygen concentration in cells

The feasibility of using EPR and the paramagnetic derivative of coal 'fusinite' to measure intracellular oxygen concentration in cultured cells in which this substance was internalized in the cytoplasm was examined. First, the possible cytotoxic effects of fusinite on cultured cells were ruled out by both morphological as well as by growth characteristics analyses. After construction of a calibration curve in which the EPR spectral linewidth of this substance was measured in response to known oxygen concentrations, the efficacy of using fusinite in the determination of intracellular oxygen concentration in cells was also tested by flowing different known oxygen gas mixtures outside cultured cells. The results indicate that fusinite is able of measuring the variations in cytoplasmic oxygen concentration that exist in response to the different gas mixtures. In addition, as an example of a possible use of fusinite, data are also presented demonstrating a decrease in cytoplasmic oxygen concentration during respiration in cells with a limited supply of oxygen. In fact, as the oxygen is consumed by the cells, the linewidth of fusinite narrows giving an intracellular oxygen concentration corresponding to zero. From the results obtained, fusinite appears to represent a new extremely precise biophysical cellular oxygen probe which may prove useful in the understanding of the complex interrelationships between oxygen and normal cell physiology and/or pathology.