Simultaneous imaging of intracellular and blood oxygen levels in tissues provides valuable information on the dynamic behavior of molecular oxygen (O₂) in normal and diseased tissues. Here, to achieve this goal, we developed green-emitting intracellular O₂ probes based on the Ir(III) complex, PPY (tris(2-phenylpyridinato)iridium(III)), and investigated the possibility of multicolor O₂ imaging by co-staining tissues with a red-emitting intravascular probe BTP-PEG₄₈. 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 O₂ 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 O₂ sensitivity in HeLa cells. Phosphorescence lifetime imaging microscopy (PLIM) measurements of HeLa cells co-stained with PPYDM and hydrophilic BTP-PEG₄₈ allowed for the evaluation of intracellular and extracellular O₂ levels in cell culture. We took PLIM images of the pancreas following intravenous administration of PPYDM and BTP-PEG₄₈ into anesthetized mice. The PLIM measurements using these probes allowed simultaneous O₂ imaging of acinar cells and capillaries in the pancreas with cellular-level resolution. From the phosphorescence lifetimes of PPYDM and BTP-PEG₄₈ 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.

Nao Matsumura

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