In vivo O2 imaging in hepatic tissues by phosphorescence lifetime imaging microscopy using Ir(III) complexes as intracellular probes

Abstract

Phosphorescence lifetime imaging microscopy (PLIM) combined with an oxygen (O₂)-sensitive luminescent probe allows for high-resolution O₂ imaging of living tissues. Herein, we present phosphorescent Ir(III) complexes, (btp)₂Ir(acac-DM) (Ir-1) and (btp-OH)₃Ir (Ir-2), as useful O₂ 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 O₂ level and distribution in spheroids, including the detection of alterations in O₂ 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 O₂ gradient in hepatic lobules with cellular-level resolution, and the O₂ levels were derived based on calibration using cultured cells; the phosphorescence lifetime of Ir-1 gave reasonable O₂ levels, whereas Ir-2 exhibited much lower O₂ levels. Intravenous administration of NH₄Cl to mice caused the hepatic tissues to experience hypoxia, presumably due to O₂ consumption to produce ATP required for ammonia detoxification, suggesting that the metabolism of the probe molecule might affect liver O₂ levels.

Figure 1

(A) Chemical structures of BTPDM1 (Ir-1) and (btp-OH)₃Ir (Ir-2). Absorption and phosphorescence spectra of Ir-1 and Ir-2 in MeCN at room temperature are shown. The phosphorescence spectra were taken in both degassed (solid line) and aerated (dashed line) solutions. (B) Schematic views of hepatic lobules. CV central vein, PV portal vein.

Figure 2

Intensity (upper) and PLIM (lower) images of HT-29 cells stained with (A) Ir-1 and (B) Ir-2 under different pO₂ in an incubator. The average phosphorescence lifetimes are shown below each image. Cells were treated with Ant A (5–21% O₂) and Na₂SO₃ (0% O₂). Scale bar: 50 µm. (C) Stern–Volmer plots of ${\tau }_{\text{p}}^0$/${\tau }_{\text{p}}$ as a function of pO₂ for Ir-1 (red) and Ir-2 (blue) partitioned into HT-29 cells under different pO₂ in an incubator. Error bars: S.D.

Figure 3

(A) Bright-field image (left) and schematic view (right) of an HT-29 cell spheroid. (B, C) Z-stacked phosphorescence intensity (left) and lifetime (right) images of an HT-29 cell spheroid stained with (B) Ir-1 and (C) Ir-2. Each image corresponds to cross-section from the bottom to the upper part at an interval of 10 µm along the z-axis. Scale bar: 100 µm. (D) Line profiles of phosphorescence lifetime (blue) and pO₂ (red) along the arrows shown in B and C. (E) Average phosphorescence lifetime (blue) and pO₂ (red) of the square region along the z-axis in an HT-29 cell spheroid stained with probes.

Figure 4

Imaging of the oxygen status of an HT-29 cell spheroid upon metabolic stimulation with FCCP or AntA. (A) Schematic representation of a spheroid in media containing FCCP or AntA. PLIM images were taken at 30 μm from the bottom after oil sealing. (B,C) Variation in PLIM images (left), and their average phosphorescence lifetime and pO₂ (right) of an HT-29 cell spheroid stained with (B) Ir-1 and (C) Ir-2 by metabolic stimulation with FCCP (1 μM) at 5 min and 6 min, respectively, and (D) Ir-1 and (E) Ir-2 by metabolic stimulation with AntA (1 μM) at 5 min. Scale bar: 100 µm.

Figure 5

PLIM images of the hepatic surface of a mouse administered (A) Ir-1 and (B) Ir-2. The color bar indicates phosphorescence lifetime in μs. Left images: 10 × objective lens, center images: 20 × objective lens, right images: 40 × objective lens. Scale bar: 200 µm (left images), 100 µm (center images), and 50 µm (right images). CV: central vein, PV: portal vein. (C, D) Phosphorescence lifetime of Ir-1 and Ir-2 and pO₂ in hepatic lobules. Average of phosphorescence lifetime and pO₂ are shown. N = 23 ROIs for CV, 23 for IR (intermediate region between CV and PV), and 24 for PV in 5 mice administered Ir-1. N = 21 ROIs for CV, 23 for MR, and 27 for PV in 5 mice administered Ir-2. *p value < 0.01 by 2-tailed unpaired t test. Error bar: S.D.

Figure 6

(A,B) Variation of PLIM images of hepatic lobules after administration of NH₄Cl following Ir-1 injection into the tail vein of a mouse. Scale bar: 200 µm. (C,D) Variation of average phosphorescence lifetime and pO₂ of ROIs in the pericentral region (ROI1) and periportal regions (ROI2 and ROI3) shown in B. Error bar: S.D.