Barometric calibration of a luminescent oxygen probe

The invention of the phosphorescence quenching method for the measurement of oxygen concentration in blood and tissue revolutionized physiological studies of oxygen transport in living organisms. Since the pioneering publication by Vanderkooi and Wilson in 1987, many researchers have contributed to the measurement of oxygen in the microcirculation, to oxygen imaging in tissues and microvessels, and to the development of new extracellular and intracellular phosphorescent probes. However, there is a problem of congruency in data from different laboratories, because of interlaboratory variability of the calibration coefficients in the Stern-Volmer equation. Published calibrations for a common oxygen probe, Pd-porphyrin + bovine serum albumin (BSA), vary because of differences in the techniques used. These methods are used for the formation of oxygen standards: chemical titration, calibrated gas mixtures, and an oxygen electrode. Each method in turn also needs calibration. We have designed a barometric method for the calibration of oxygen probes by using a regulated vacuum to set multiple PO2 standards. The method is fast and accurate and can be applied to biological fluids obtained during or after an experiment. Calibration over the full physiological PO2 range (1-120 mmHg) takes ∼15 min and requires 1-2 mg of probe.