Intracellular Oxygen Sensing Using Long-Phosphorescence-Lifetime Cationic Ir(III) Complexes With Coumarin 6 and 545T Ligands

Real-time monitoring of intracellular oxygen (O₂) is crucial for understanding cell proliferation, differentiation, and metabolic processes, as well as for observing cellular behavior within hypoxic tissues associated with conditions such as cancer, ischemia, and chronic kidney disease. We developed small-molecule intracellular O₂ probes based on the cationic Ir(III) complexes C6-BA and C545T-BA (C6 = coumarin 6, C545T = coumarin 545T, and BA = <i>N,N'</i>-dibutylethylenediamine) and investigated intracellular O₂ levels using a commercially available microplate reader and phosphorescence lifetime imaging microscope (PLIM). The synthesized Ir(III) complexes exhibited a strong visible absorption band with a molar absorption coefficient of approximately 100,000 dm^-3 mol^-1 cm^-1 due to singlet ligand-centered π,π* transitions originating from the C6 and C545T ligands. C6-BA and C545T-BA exhibited bright emission with high quantum yield (0.65 for C6-BA, 0.44 for C545T-BA) and remarkably long emission lifetimes (34.8 μs for C6-BA, 53.7 μs for C545T-BA) in N₂-saturated acetonitrile. A comprehensive study of cellular analyses of these complexes revealed that C6-BA and C545T-BA are efficiently taken up by cultured cells and exhibit high O₂ sensitivity. The phosphorescence lifetime of living cells stained with each Ir(III) complex determined by measuring time-resolved emission using a microplate reader enabled tracking of real-time changes in cellular O₂ tension after treatment with metabolic stimulants. Furthermore, we acquired PLIM images of living cells stained with C6-BA followed by treatment with metabolic stimulants. Time-lapse PLIM analysis showed a heterogeneous O₂ distribution within the field of view and variations in O₂ partial pressure between individual cells.