Femtosecond three-photon excitation and single-photon timing detection of α-NPO fluorescence

We demonstrate the application of three-photon excitation to fluorescence probe studies using time-correlated single-photon counting (TCSPC). By exciting with 120 fs Ti:sapphire laser pulses at 800 nm we have observed fluorescence emission from the scintillator 2-(1-napthyl)-5-phenyloxazole (α-NPO) in solutions and small unilamellar vesicles (SUVs) of L-α-dipalmitoylphosphatidylcholine (DPPC). In SUVs the time-resolved excimer emission and fluorescence anisotropy are consistent with a heterogeneous distribution of α-NPO molecules between isolated sites and ground state clusters in a similar manner to that which we reported previously for 2,5-diphenyloxazole (PPO).