Time-resolved stimulated emission depletion and energy transfer dynamics in two-photon excited EGFP

Time and polarization-resolved stimulated emission depletion (STED) measurements are used to investigate excited state evolution following the two-photon excitation of enhanced green fluorescent protein (EGFP). We employ a new approach for the accurate STED measurement of the hitherto unmeasured degree of hexadecapolar transition dipole moment alignment ⟨α40⟩ present at a given excitation-depletion (pump-dump) pulse separation. Time-resolved polarized fluorescence measurements as a function of pump-dump delay reveal the time evolution of ⟨α40⟩ to be considerably more rapid than predicted for isotropic rotational diffusion in EGFP. Additional depolarization by homo-Förster resonance energy transfer is investigated for both ⟨α20⟩ (quadrupolar) and ⟨α40⟩ transition dipole alignments. These results point to the utility of higher order dipole correlation measurements in the investigation of resonance energy transfer processes