Novel fluorescent oxygen indicator for intracellular oxygen measurements

Intracellular oxygen concentration is of primary importance in determining numerous physiological and pathological processes in biological systems. In this paper, we describe the application of the oxygen sensing indicator, ruthenium dibipyridine 4-(1"-pyrenyl)-2,2'-bipyridine chloride [Ru(bpy-pyr)(bpy)(2)], for molecular oxygen measurement in J774 murine macrophages. Ru(bpy-pyr)(bpy)(2) exhibits strong visible absorption, efficient fluorescence, long excited state lifetime, large Stokes shift, and high photo- and chemical stability. The fluorescence of Ru(bpy-pyr)(bpy)2 is efficiently quenched by molecular oxygen. It is 13 fold higher in a nitrogenated solution than in an oxygenated one. The dye passively permeates into cells and maintains its oxygen sensitivity for at least 5 h when the cells are stored in a phosphate buffered saline solution at pH 7.4. The oxygen sensitivity, photostability, and chemical stability of the indicator and the effect of hypoxia and hyperoxia on the intracellular oxygen level in single macrophages are discussed.