Fig. 3: Phototoxicity assessment during a fluorescence live-cell imaging acquisition.

The PhotoFiTT workflow was tested using the fluorescent probe MitoTracker. a Acquisition experimental design using PhotoFiTT. Live-cell imaging of MitoTracker-stained cells (green FM and CMXRed) exposed to 475 nm fluorescence excitation light (adjusted to 20 min; green) before releasing the CDK1 inhibitor drug and allowing the cells to enter mitosis. During acquisition, transmitted light (grey) was used to image brightfield. b Quantification of cell rounding delays across different MitoTracker probes. The control population peaks at t 50 min, representing the average cell rounding time. Exposure to 475 nm light induces a delay that becomes more apparent when MitoTracker Green FM is excited with the right light wavelength. c Temporal analysis of mother and daughter cell populations across 475 nm light doses. d Cumulative cell activity over 7 h post-exposure normalised for each replica (logarithmic scale). The synchronised population presented physiological behaviour while both Mitotracker CMXRed and Green FM depicted changes in activity. N values correspond to each acquired field of view. Excitation at 475 nm (Unlabelled (n = 9), MitoTracker Red CMXRos (n = 20), MitoTracker Green FM (n = 20), MitoTracker Red CMXRos excited at 475 nm (n = 20) and MitoTracker Green FM excited at 475 nm (n = 20)). A total of two biological replicates with ten technical replicates were done In (b–d) data were represented as mean values ± 95% confidence interval. The boxes in (c) show the quartiles of the dataset while the whiskers extend to the entire distribution, except for distributions where outliers surpass the inter-quartile range. Statistical significance estimated by Kolmogorov–Smirnov hypothesis test with * (p value <0.05), ** (p value <0.01), *** (p value <0.001), **** (p value <0.0001).