Fig. 3. Multiplexed imaging of cellular superoxide levels with probes F-Tz1 and F-Tz4.

A, B Fluorescence images of HepG2 cells stained with either F-Tz1 or F-Tz4 (each 5 μM, 30 min). Cells were pretreated with various doses of H₂O₂ for 2 h (A) or 2 mM H₂O₂ for various time (B). Scale bar: 25 μm. C, D The statistically quantified data on the cellular fluorescence intensity in (A) and (B). The data were normalized to the control group, and P values were analyzed by two-tailed unpaired t-test, 95% Confidence interval. F-Tz1: n = 97 cells for 0 mM H₂O₂ 0 h, n = 45 cells for 0.5 mM H₂O₂ 2 h, n = 66 cells for 1 mM H₂O₂ 2 h, n = 49 cells for 2 mM H₂O₂ 2 h, n = 56 cells for 2 mM H₂O₂ 1 h, n = 41 cells for 2 mM H₂O₂ 4 h; F-Tz4: n = 116 cells for 0 mM H₂O₂ 0 h, n = 78 cells for 0.5 mM H₂O₂ 2 h, n = 68 cells for 1 mM H₂O₂ 2 h, n = 77 cells for 2 mM H₂O₂ 2 h, n = 68 cells for 2 mM H₂O₂ 1 h, n = 74 cells for 2 mM H₂O₂ 4 h. All cell numbers are over three biologically independent experiments. E, F Fluorescence images of HepG2 cells co-stained with F-Tz4 (5 μM, 30 min) and various organelle markers (50 nM Mito Tracker Red CMXRos, 50 nM Lyso Tracker Red, or 5 μM DHE for nuclei, 15 min). Cells were pretreated either with a low (0.5 mM) or high (2 mM) dose of H₂O₂ for 2 h before being stained with the probes. The right panel showed the intensity profile and PCC (Pearson’s correlation coefficient) along the white rectangle highlighted in the left panel. Scale bars, 10 μm.