Background Of the few vital DNA and RNA probes, the SYTO dyes are the most specific for nucleic acids. However, they show no spectral contrast upon DNA or RNA binding. We show that fluorescence lifetime imaging using two-photon excitation of SYTO13 allows differential and simultaneous imaging of DNA and RNA in living cells, as well as sequential and repetitive assessment of staining patterns. Methods: Two-photon imaging of SYTO13 is combined with lifetime contrast, using time-gated detection. We focus on distinguishing DNA and RNA in healthy and apoptotic Chinese hamster ovary cells. Results: In healthy cells, SYTO13 has a fluorescence lifetime of 3.4 +/- 0.2 ns when associated with nuclear DNA. Bound to RNA. its lifetime is 4.1 +/- 0.1 ns. After induction of apoptosis, clusters of SYTO13 with fluorescence lifetime of 3.4 +/- 0.2 ns become apparent in the cytoplasm. They are identified as mitochondrial DNA on the basis of colocalization experiments with the DNA-specific dye, DRAQ5, and the mitochondrial-specific dye, CMXRos. Upon progression of apoptosis, the lifetime of SYTO13 attached to DNA shortens significantly, which is indicative of changes in the molecular environment of the dye. Conclusions: We have characterized SYTO13 as a vital lifetime probe, allowing repetitive and differential imaging of DNA and RNA.