Fig. 2: Quantitative phototoxicity assessment with PhotoFiTT.

a PhotoFiTT integrates biochemical experiments, microscopy acquisition, and image analysis. Cell synchronisation is induced by CDK1 inhibition (RO-3306), arresting cells at the G2/M interface. Synchronised cells are washed prior to 6-h time-lapse imaging, including transmitted light and fluorescence markers for model training. Cell rounding is identified using trained models to quantify cell activity and division dynamics. Detailed workflow descriptions are in Notes S1 and S2. b Time-lapse brightfield imaging of an adherent mammalian cell (CHO Chinese hamster ovary) undergoing mitosis. The mother cell’s diameter approximately doubles that of the resulting daughter cells post-division. Scale bar 20 µm. c Temporal distribution of mitotic cell rounding in synchronised populations exposed to varying doses of 385 nm (near-UV) light J/cm²). Exposed populations exhibit a dose-dependent delay in cell rounding, manifested as rightward shifts in the distribution peaks. d Quantification of cell rounding delays across different light wavelengths and doses. The control population peaks at 50 min, representing the average cell rounding time (indicated with a line in b). Exposure to 385 nm light induces a dose-dependent delay, while 475 and 630 nm wavelengths only cause significant delays at high doses (60 J/cm²). e Cell volume nearly halves as mother cells divide into two daughter cells. Low-dose exposure (0.6 J/cm²) delays this transition, while high doses result in heterogeneous populations at later time points (120 min), indicating asynchronous divisions and potential cell cycle arrest. f Temporal analysis of mother and daughter cell populations across different wavelengths and doses. All wavelengths induce delays in daughter cell appearance, with 385 nm light and high doses of 475 and 630 nm light causing the most pronounced effects. g Cumulative cell activity over 7 h post-exposure normalised for each replica (logarithmic scale). All light exposures reduce overall cell activity, with 385 and 475 nm showing more dose-dependent effects than 630 nm light. These results collectively demonstrate the wavelength- and dose-dependent impacts of light exposure on cell division dynamics and overall cellular activity. N values correspond to each acquired field of view. In 385 nm (0 (n = 130), 0.6 (n = 44), 6 (n = 22), and 60 (n = 34)), 475 nm (0 (n = 80), 0.6 (n = 25), 6 (n = 15), and 60 (n = 5)), and 630 nm (0 (n = 57), 0.6 (n = 39), 6 (n = 38), and 60 (n = 16)). A total of five biological replicates with ten technical replicates were done. In d, g data were represented as mean values ± 95% Confidence Interval. The boxes in (b) 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 two-sided Kolmogorov–Smirnov hypothesis test with * (p value <0.05), ** (p value <0.01), *** (p value <0.001), **** (p value <0.0001). Details and exact statistical values available in the Supplementary Information.