Oxygen-dependent quenching of phosphorescence: Quantitating and improving in vivo oxygen measurements

The goal of this research was to quantitate and extend oxygen measurements in the tissue of living organisms using the oxygen dependent quenching of phosphorescence. This method uses light to determine the oxygen pressure in media in which an appropriate phosphor is incorporated, including biological fluids, quantitatively, non-invasively, and with excellent temporal resolution (milliseconds). A thermostated calibration system was developed for precise chemical titration of the oxygen in solution while making simultaneous measurements of phosphorescence lifetime and of oxygen pressure with a high accuracy oxygen electrode. Several phosphors, including a Pd-tetrabenzoporphyrin (Green 2W) which both absorbs and phosphoresces in the near infra red region of the spectrum, were calibrated. Green 2W was then used to obtain 2 dimensional images of the oxygen distribution in the tissue of mice with subcutaneous EMT6 tumors. These measurements were made through the intact skin and, with trans-illumination, through the thickness of the body. They show that the oxygen pressure was much lower in the tumors than in the surrounding normal tissue. A series of phosphors specifically designed for use in biological systems, i.e. water soluble, excreted by the kidneys, and without tissue toxicity, were calibrated and then tested in vivo. One of these, Oxyphor R2, was shown to provide excellent oxygen measurements and to meet the above criteria. A micro-light guide phosphorometer was developed for measuring oxygen pressure at local points at precise depths in tissue. The excitation light was sinusoidally modulated and a phase lock amplifier used to measure the phosphorescence lifetime by the phase shift between the excitation and phosphorescence light. The excitation light was conducted to the tissue through an aluminum coated 200 $\mu$m optical fiber. This fiber was surrounded by a bundle of 15 fibers, each 50 $\mu$m in diameter, for collecting the emitted phosphorescence. The complete microlight guide was contained in a sharpened stainless steel needle, 400 $\mu$m outside diameter. The oxygen levels in the striatum of the brain of anesthetized newborn piglets, a structure 1.5 cm below the cortical surface, and its dependence on oxygen pressure in the inspired gas was measured