Use of a fluorescence lifetime imaging microscope in an apoptosis assay of Ewing's sarcoma cells with a vital fluorescent probe

A fluorescence lifetime imaging microscope (FLIM) was applied to study early-stage apoptotic cells stained with a SYTO13 dye. The fluorescence lifetime of SYTO13 in healthy cells was 3.8 ± 0.3 ns but was reduced to 2.4 ± 0.4 and 1.9 ± 0.2 ns after a 3-h period of incubation with SYTO13 when doxorubicin, a known inducer of apoptosis, was added to human Ewing's family tumor cells at final concentrations of 250 and 500 nM, respectively, in a dose-dependent experiment. On the other hand, in a time-dependent experiment, the fluorescence lifetime decreased to 2.5 ± 0.5 and 1.7 ± 0.4 ns at a doxorubicin concentration of 750 nM after 2 and 4 h, respectively. A possible explanation for these results is self-quenching induced by a change in interprobe distance that arises from the condensation of DNA during apoptosis. In this study, the FLIM system was employed to investigate early-stage apoptosis that involves only small morphological changes, suggesting the potential advantage of this method for evaluating small biological effects in living cells.