Fast excitation-driven fluctuations in the fluorescence emission of yellow-shifted green fluorescent protein mutants T203Y and T203F, with S65G͞S72A, are discovered in the 10 ؊6 -10 ؊3 -s time range, by using fluorescence correlation spectroscopy at 10 ؊8 M. This intensity-dependent flickering is conspicuous at high pH, with rate constants independent of pH and viscosity with a minor temperature effect. The mean flicker rate increases linearly with excitation intensity for at least three decades, but the mean dark fraction of the molecules undergoing these dynamics is independent of illumination intensity over Ϸ6 ؋ 10 2 to 5 ؋ 10 6 W͞cm 2 . These results suggest that optical excitation establishes an equilibration between two molecular states of different spectroscopic properties that are coupled only via the excited state as a gateway. This reversible excitation-driven transition has a quantum efficiency of Ϸ10 ؊3 . Dynamics of external protonation, reversibly quenching the fluorescence, are also observed at low pH in the 10-to 100-s time range. The independence of these two bright-dark flicker processes implies the existence of at least two separate dark states of these green fluorescent protein mutants. Time-resolved fluorescence measurements reveal a single exponential decay of the excited state population with 3.8-ns lifetime, after 500-nm excitation, that is pH independent. Our fluorescence correlation spectroscopy results are discussed in terms of recent theoretical studies that invoke isomerization of the chromophore as a nonradiative channel of the excited state relaxation.

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This paper reports spectroscopic investigations of the chromophore terrylene embedded in a matrix of crystalline p-terphenyl. While this system is particularly well suited for single molecule spectroscopy, little is known about the guest site configuration of terrylene. To shed some light on this issue, we employed absorption and fluorescence spectroscopy and compared the experimental data to the results of theoretical calculations. Based on this comparison we suggest a substitution scheme which is in agreement with all the spectroscopic evidence. The dispersed fluorescence spectra of single molecules in the wings of the inhomogeneous distribution deviate significantly from the bulk spectra. This observation is discussed in terms of a host-induced change of the structure and a possible C13 isotopic substitution of the chromophores. Finally we investigated the dynamic host–guest interactions via the temperature-dependent shift and broadening of single molecule excitation lines and found these processes dominated by coupling to characteristic pseudolocal phonon modes of the host, although chromophores in the wings of the inhomogeneous distribution exhibit additional contributions which we attribute to thermal matrix expansion.