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Simultaneous imaging of intracellular and blood oxygen levels in tissues provides valuable information on the dynamic behavior of molecular oxygen (O 2 ) in normal and diseased tissues. Here, to achieve this goal, we developed green-emitting intracellular O 2 probes based on the Ir(III) complex, PPY (tris(2-phenylpyridinato)iridium(III)), and investigated the possibility of multicolor O 2 imaging by co-staining tissues with a red-emitting intravascular probe BTP-PEG 48 . The newly synthesized complexes possess modified 2-phenylpyridinato ligand(s) with a cationic or hydrophilic substituent, such as a dimethylamino group, triphenylphosphonium cation, or hydroxy group, in order to enhance cellular uptake efficiency. The photophysical and cellular properties of these complexes were systematically investigated to evaluate their ability as O 2 probes. Among these complexes, PPYDM and PPY2OH, which have a dimethylamino group and two hydroxy groups, respectively, exhibited much higher cellular uptake efficiencies compared with PPY and showed high O 2 sensitivity in HeLa cells. Phosphorescence lifetime imaging microscopy (PLIM) measurements of HeLa cells co-stained with PPYDM and hydrophilic BTP-PEG 48 allowed for the evaluation of intracellular and extracellular O 2 levels in cell culture. We took PLIM images of the pancreas following intravenous administration of PPYDM and BTP-PEG 48 into anesthetized mice. The PLIM measurements using these probes allowed simultaneous O 2 imaging of acinar cells and capillaries in the pancreas with cellular-level resolution. From the phosphorescence lifetimes of PPYDM and BTP-PEG 48 and the calibration curves evaluated in rat pancreatic acinar cells and blood plasma, we found that the average oxygen partial pressures of acinar cells and capillaries were almost equal at about 30 mmHg.