Fluorescence lifetime imaging of oxygen in living cells

The usefulness of the fluorescent probe ruthenium tris(2,2′-dipyridyl) dichloride hydrate (RTDP) for the quantitative imaging of oxygen in single cells was investigated utilizing fluorescence life-time imaging. The results indicate that the fluorescence behavior of RTDP in the presence of oxygen can be described by the Stern-Volrner equation. This shows that fluorescence quenching by oxygen is a dynamic quenching process. In addition, it was demonstrated that the fluorescence lifetime of RTDP is insensitive to pH, ion concentration, and cellular contents. This implies that a simple calibration procedure in buffers can be used to quantify oxygen concentrations within cells. First fluorescence imaging experiments on J774 macrophages show a nonuniform fluorescence intensity and a uniform fluorescence lifetime image. This indicates that the RTDP is heterogeneously partitioned throughout the cells, while the oxygen concentration is constant. © 1997 Plenum Publishing Corporation.