In vivo O imaging in hepatic tissues by phosphorescence lifetime imaging microscopy using Ir(III) complexes as intracellular probes.

Phosphorescence lifetime imaging microscopy (PLIM) combined with an oxygen (O)-sensitive luminescent probe allows for high-resolution O imaging of living tissues. Herein, we present phosphorescent Ir(III) complexes, (btp)Ir(acac-DM) (Ir-1) and (btp-OH)Ir (Ir-2), as useful O probes for PLIM measurement. These small-molecule probes were efficiently taken up into cultured cells and accumulated in specific organelles. Their excellent cell-permeable properties allowed for efficient staining of three-dimensional cell spheroids, and thereby phosphorescence lifetime measurements enabled the evaluation of the O level and distribution in spheroids, including the detection of alterations in O levels by metabolic stimulation with an effector. We took PLIM images of hepatic tissues of living mice by intravenously administrating these probes. The PLIM images clearly visualized the O gradient in hepatic lobules with cellular-level resolution, and the O levels were derived based on calibration using cultured cells; the phosphorescence lifetime of Ir-1 gave reasonable O levels, whereas Ir-2 exhibited much lower O levels. Intravenous administration of NHCl to mice caused the hepatic tissues to experience hypoxia, presumably due to O consumption to produce ATP required for ammonia detoxification, suggesting that the metabolism of the probe molecule might affect liver O levels.