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.

Intracellular oxygen concentration is of primary importance in determining numerous physiological and pathological processes in biological systems. This paper describes the development and application of micrometer-sized oxygen-sensitive fluorescence lipobeads for intracellular measurements of molecular oxygen in J774 murine macrophages. A ruthenium diimine complex [Ru(bpy-pyr)(bpy)2]C12 (bpy = 2,2'-bipyridine, bpy-pyr = 4-(1"-pyrenyl)-2,2'-bipyridine) is used as the oxygen indicator. The indicator exhibits high chemical and photostability and high sensitivity to oxygen. The indicator molecules are immobilized in a phospholipid membrane that coats polystyrene microparticles. The fluorescence of the lipobeads is effectively quenched by molecular oxygen. The fluorescence intensity of the oxygen-sensitive lipobeads is 3 times higher in a nitrogenated solution than in an oxygenated solution. The lipobeads are internalized by murine macrophages through phagocytosis. They maintain their spectral properties for 24 h in living cells when the cells are stored in phosphate-buffered saline at pH 7.4. The photostability, reversibility, and effect of hypoxia, hyperoxia, and oxidative stress on the intracellular level of oxygen in J774 murine macrophages are described.

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