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Measurement of intracellular oxygen concentration using the spin label TEMPOL

We have developed a noninvasive method with general applicability for measuring intracellular oxygen using the spin label TEMPOL (2, 2, 6, 6‐tetramethypiperidine‐N‐oxyl‐4‐ol) which has superhyperfine structure in its electron spin resonance spectra that is broadened in the presence of oxygen. This broadening is linear over a range of 1 to 6 ppm oxygen which covers the important physiological range of oxygen concentration. Viscosity, TEMPOL concentration, and instrument modulation intensity also can affect superhyperfine structure but the contributions from these effects can be determined. The TEMPOL distributes equally into the intra‐and extracellular compartments but its intracellular signal can be studied selectively by addition of transition metal ions such as potassium ferricyanide and potassium tris (oxalato)chromiate, which broaden away the signal from extracellular TEMPOL and do not cross the cell membrane to affect the intracellular TEMPOL Results with a cell culture line (mouse thymus‐bone marrow) indicate that under our experimental conditions these cells may maintain an average intracellular oxygen concentration lower than the extracellular oxygen concentration, and that there is not a constant relationship between extracellular and intracellular oxygen concentrations.