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

Abstract

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 r 0 of PPO in glycerol at −5 °C measured for twophoton excitation is significantly higher than that observed for one-photon excitation. The r 0 value of 0.54 for two-photon excitation is well in excess of the theoretical maximum of 0.4 for singlephoton excitation. A similar value of r 0 ≃ 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 r 0 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.

Two-photon fluorescence: Resonance energy transfer

“…These results, which reproduce those derived elsewhere, [10][11][12][13]26,27 are also based on the assumption that rotational relaxation is slow compared to the fluorescence lifetime, as commonly applies for large chromophores in viscous media, and also as justified by many experimental results. [10][11][12][13] Thus Eqs. ͑5͒, ͑7͒-͑11͒ model the incoherent ultrafast molecular response of the system.…”

“…Other multiphoton fluorescence studies that have been reported relate directly to biological and other systems featuring both single center chromophores and energy trapping sites. [10][11][12][13][14][15][16][17][18][19][20] These, together with the well-known use of FRET as a spectroscopic ruler, [21][22][23][24][25] reflect the practical utility of the theory to be developed.…”

“…The limiting results have previously been derived by others, 11,12,26,27 but are included here for the initial development of the more general theory. The results are then extended to a two-chromophore system in which the twophoton energy at the donor site is transferred via resonant energy transfer, subject to nonradiative losses, to an acceptor species from which the signal radiation then emerges.…”

Time-resolved two-photon induced anisotropy decay: The rotational diffusion regime

“…Figure I shows plots of R(O) and R(oo) as a function of C with P=O. 5. The values of R(O) and R(oo) are distinct except at the point C=YJ, which occurs for an oblate symmetric rotor (with Figure 2 shows plots of the time dependence R(t) for (a) C=-l and P=0.5, and for (b) C=-l and P=-0.5.…”

Photoselection of Luminescent Molecules in Anisotropic Media in the Case of Two-Photon Excitation. Part I. Theoretical Considerations

“…As recently demonstrated, a markedly higher steady-state limiting anisotropy can be observed in two-photon than in one-photon excitation [10][11][12] two-and three-photon excitation are 4/7 and 2/3, re spectively, compared to 2/5 measured upon one-photon excitation [12]. In this case, the higher limiting emission anisotropy is indicative of a better degree of orientation due to photoselection.…”