Phosphorescent soft salt for ratiometric and lifetime imaging of intracellular pH variations

Abstract

In contrast to traditional short-lived fluorescent probes, long-lived phosphorescent probes based on transition-metal complexes can effectively eliminate unwanted background interference by using time-resolved luminescence imaging techniques, such as photoluminescence lifetime imaging microscopy. Hence, phosphorescent probes have become one of the most attractive candidates for investigating biological events in living systems. However, most of them are based on single emission intensity changes, which might be affected by a variety of intracellular environmental factors. Ratiometric measurement allows simultaneous recording of two separated wavelengths instead of measuring mere intensity changes and thus offers built-in correction for environmental effects. Herein, for the first time, a soft salt based phosphorescent probe has been developed for ratiometric and lifetime imaging of intracellular pH variations in real time. Specifically, a pH sensitive cationic complex (C1) and a pH insensitive anionic complex (A1) are directly connected through electrostatic interaction to form a soft salt based probe (S1), which exhibits a ratiometric phosphorescent response to pH with two well-resolved emission peaks separated by about 150 nm (from 475 to 625 nm). This novel probe was then successfully applied for ratiometric and lifetime imaging of intracellular pH variations. Moreover, quantitative measurements of intracellular pH fluctuations caused by oxidative stress have been performed for S1 based on the pH-dependent calibration curve.

Fig. 1. Design concept of a ratiometric pH probe and chemical structures of complexes A1, C1 and S1.

Fig. 2. (a) Normalized absorption and photoluminescence spectra of A1 and C1 in acetonitrile solution. (b) Photoluminescence spectra of S1 at different concentrations in acetonitrile solution. (c) Photoluminescence spectra of anionic complex A1 (10^–5 M) in acetonitrile solution with various amounts of cationic complex C1 (0–1.0 × 10^–5 M). (d) Stern–Volmer plot of the quenching study between C1 and A1 ([Q] is the concentration of quencher).

Fig. 3. (a) Changes in the phosphorescence emission spectra of A1 (2.0 × 10^–5 M) in the pH range of 2.03–7.94 in CH₃CN/buffer (1 : 9, v/v). (b) Changes in the phosphorescence emission spectra of C1 (2.0 × 10^–5 M) in the pH range of 2.03–7.94 in CH₃CN/buffer (1 : 9, v/v). (c) Changes in the phosphorescence emission spectra of S1 (2.0 × 10^–5 M) in the pH range of 2.03–7.94 in CH₃CN/buffer (1 : 9, v/v). (d) Plot of I_{625 nm}/I_{451 nm}versus pH values. I_{625 nm} and I_{451 nm} indicate the phosphorescence intensity at 625 nm and at 451 nm, respectively.

Fig. 4. (a) Living HepG-2 cells co-stained with 10 μM A1 and C1 for 1 h at 37 °C, and (b) living HepG-2 cells incubated with S1 under the same conditions.

Fig. 5. (a) Phosphorescence images of S1 in HepG-2 cells clamped at pH 3.98, 5.02, 6.08, 7.01 and 8.01, respectively. The excitation wavelength was 405 nm and the images of the first row (blue channel) and second row (red channel) were collected in the ranges of 430–480 nm and 600–700 nm, respectively. Overlay images (third row) and ratio images obtained from the red and blue channels (fourth row). (b) Phosphorescence emission spectra of the HepG-2 cells at pH 3.98 and 8.01.

Fig. 6. Phosphorescence lifetime images of S1 in living HepG-2 cells at different pH values. HepG-2 cells were incubated for 1 h at 37 °C.

Fig. 7. (a) Intracellular pH calibration curve of S1 in HepG-2 cells. (b) Ratiometric images and (c) phosphorescence lifetime images of S1 (10 μM). Intact cells, H₂O₂ (100 μM) treated, NEM (100 μM) treated and NAC (100 μM) treated cells were incubated for 1 h at 37 °C.