Figure 6

Increase in mitochondrial oxidative stress at increasing 2p-laser power excitation in HeLa cells. Representative confocal images of MitoSOX (mitochondrial ROS/superoxide) signal imaged under identical conditions with 514 nm at 10% (Em-551–683 nm, PMT 750v), after FLIM acquisition with 740 nm. All images were contrasted for brightness at the same settings to show the expected low-level ROS signal in cancer cells in the control image. Confocal MitoSOX signal after FLIM at laser average power of (a) 5.47 mW-Control and after increased FLIM laser power of (b) 12.88 mW and (c) 19.21 mW. The rising MitoSOX signal is indicative of oxidative stress not apparent in control (a). Hoechst nuclear stain was applied for nuclear localization. (d) Mean intensity (± SEM) bar plots and mean ROIs counts (2 × 2 pixel, orange line) from 4 FOVs show increase in MitoSOX Red mean gray-level intensity (8-bit depth) from 17.64 to 19.87 (12.6% increase) to 32.52 (84.3% increase); and mean ROI counts (orange line) from 10 to 427 (~ 40-fold increase) to 2964.75 (~ 300 fold increase) in ROI counts post FLIM with 5.47 mW-Control, 12.88 mW and 19.21 mW respectively-indicating increase in oxidative stress signal with higher laser average power. Single factor Anova analysis was done for mean intensity for statistical significance between groups at alpha value of 0.05. The results were statistically significant with p value of < 0.0001. Scale bar 20 μm.