Barometric Calibration of Luminescent Oxygen Probes

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, 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 inter‐laboratory variability of the calibration coefficients in the Stern ‐ Volmer equation. Published data (9 sources) for a common oxygen probe Pd‐porphyrin + BSA have CV = 9.6%, due to differences in the calibration techniques used. These are methods for the formation of oxygen standards via chemical titration, calibrated gas mixtures and an oxygen electrode. Each method in turn also needs calibration. We have designed a barometric method for calibration of oxygen probes by using a regulated vacuum chamber and manometer to set multiple PO 2 standards. The method is fast, accurate and can be applied to biological fluids sampled during or after an experiment. Calibration over the full physiological PO 2 range (1–120 mmHg) takes about 15 min and requires 1–2 mg of probe. A sample of probe solution was placed on a cotton membrane inside an evacuated vial. The vial was fixed in a thermostated (37±0.1 °C) well with the membrane in a mid‐plane position, at the intersection of the perpendicular excitation and emission optical pathways. The probe was excited by a brief light pulse (1μs duration, 410 nm) from a high power diode. The emitted phosphorescence (620–750 nm) was detected by a Hamamatsu Photosensor module and recorded by a computer at 500 kS/s with 12‐bit resolution. The excitation flash rate was 100 s −1 and 1000 decay curves were collected for 10 s for each PO 2 . Standard PO 2 's in a vial were created by a vacuum pump, equipped with a regulator and a pressure filter and was set by a precision digital manometer (PO 2 error 0.1 mmHg). The necessary correction was made for atmospheric pressure. When the pressure was changed to a higher or lower PO 2 standard, the probe in the membrane equilibrated with the air in the vial with a time constant of 1.5 s, so a 10 s time interval was used before starting data acquisition. Recorded phosphorescence decay curves were analyzed with non‐linear fitting to two different models of decays to determine the exponential decay constants. It turned out that, even in the absence of oxygen gradients and at PO 2 = 0, the phosphorescence decay of conjugated Pd‐porphyrin was heterogeneous, and it was fitted well by a heterogeneous model. Application of a monoexponential model was possible only for the initial segment of the decay curve or for its tail. This property did not introduce a distortion in the PO 2 measurement, if there is no “fitting delay.” A heterogeneous model fit the entire curve and was not sensitive to a delay. Thus, for consistency the experimental and calibration decay curves must be processed by exactly the same procedure. The equation of translation between data obtained with different calibration coefficients was derived for an accurate comparison of results from different laboratories.