Feasibility of single-cell tracking and the effect of repeated fluorescence excitation on survival and proliferation of Fucci-mNSC in vitro. A) An example pedigree map showing cell divisions or death within the fluorescence imaged, randomly selected Fucci-mNSC population over the course of 7 day experiment. During repeated fluorescence image acquisition, the single-cell tracked Fucci-mNSC exhibit B) decreased cell survival, but C) no significant change in the proportion of parent NSC that divide during the experiment was detected. However, D) the average number of cell divisions per parent Fucci-mNSC was decreased when compared to the control cells imaged with DIC only during a 7 day in vitro experiment. Student’s 2-tailed t-test, *p ≤ .01, **p ≤ .008, n = 4 independent biological experimental replicates, mean ± error bars SEM. D) The number of Fucci-mNSC exposed to repeated fluorescence excitation remained unchanged during a 7 day experiment relative to 0 h timepoint, while the DIC-only control group exhibited increasing number of cells towards the end of the experiment. Black brackets 1-way ANOVA **p ≤ .002, grey brackets Tukey’s t-test **p ≤ .002, n = 4 independent biological experimental replicates, mean ± error bars SEM.