Probing cell metabolism using the two-photon excitation autofluorescence lifetime of tryptophan

Compared to intensity detection, fluorescence lifetime has the advantage of being unaffected by variations in excitation intensity, fluorophore concentration, or attenuation due to biological absorption and scattering. In this Letter, to the best of our knowledge, we present the use of the two-photon excitation autofluorescence lifetime imaging of tryptophan (TRP) to probe cell metabolism for the first time. Tests of pure chemical samples showed that the fluorescence lifetime of TRP was highly sensitive to changes in molecular conformation and the environment. In in vitro cell experiments, we successfully utilized the fluorescence lifetime of TRP to distinguish tumor cells from healthy cells, track the therapeutic effect of the tumor immunotherapy drug 1-MT for HeLa cells, and monitor cells in response to carbonyl cyanide 3-chlorophenylhydrazone (CCCP)-induced cell apoptosis. These results reveal that the two-photon excitation autofluorescence lifetime of TRP could be a sensitive natural probe of cell metabolism in living cells.