Time-resolved fluorescence intensity and anisotropy decays of 2,5-diphenyloxazole by two-photon excitation and frequency-domain fluorometry

We report the first time-resolved fluorescence measurements of the intensity and anisotropy decays resulting from two-photon excitation. A 10-GHz frequency-domain fluorometer (Rev. Sci. Instrum 1990, 61, 2331), equipped with two focal lenses and an emission monochromator, was used for steady-state and time-resolved measurements of PPO fluorescence. The emission spectra and the intensity decays observed with single- and two-photon excitation were essentially identical. The steady-state limiting anisotropy r₀ of PPO in glycerol at -5°C measured for two-photon excitation is significantly higher than that observed for one-photon excitation. The r₀ value of 0.54 for two-photon excitation is well in excess of the theoretical maximum of 0.4 for single-photon excitation. A similar value of r₀ ≃ 0.50 was obtained from the frequency-domain anisotropy data with two-photon excitation of PPO in methanol, butanol, and propylene glycol at 20°C. These higher values of r₀ indicate that two-photon excitation results in a more highly oriented photoselected population, which can increase the resolution of rotational correlation times and/or complex anisotropy decays. The anisotropy resolution can still be increased by using global analysis of anisotropy decays measured with single- and two-photon excitation.