In moderately polar and viscous solvents, the emission spectra of fluorophores often shift to longer wavelengths as the excitation wavelength is increased toward the long-wavelength (red) side of the absorption. Red shifts occur because long-wavelength excitation results in photoselection of those fluorophores which are interacting most strongly with the polar solvent molecules. The observation of excitation red shifts requires that these enhanced dipole-dipole interactions are maintained in the photoselected population during the lifetime of the excited state. Consequently, the magnitude of the excitation red shifts depends upon the dynamic properties of the environment surrounding the fluorophore, as well as upon the solvent polarity and the sensitivity of the fluorophore to the polarity of the solvent. We used this phenomenon to investigate the dynamic properties of reference solvents, model membranes, and the protein apomyoglobin labeled with 6-(p-toluidinyl)-2-naphthalenesulfonic acid (TNS). The spectral shifts and lifetime data indicate that red-edge excitation results in the selective excitation of "solvent-relaxed" fluorophores. By comparison of the data obtained for TNS in solvents and bound to the macromolecules, one may estimate the relaxation rate of the environment. This comparison indicates rapid spectral relaxation of TNS bound to lipid vesicles and a somewhat slower relaxation around TNS bound to the heme site of apomyoglobin.

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Phase sensitive emission spectra recorded at multiple frequencies were used to determine the lifetimes and steady-state spectra of the fluorophores In two-component mixtures. This analysis does not require any previous knowledge of the lifetimes, fractional Intensities, or the emission spectra and is thus an Improvement over single frequency phase sensitive detection which requires known emission spectra or known lifetimes. Phase sensitive emission spectra are recorded at several frequencies and arbitrarily chosen detector phase angles. The data are analyzed by use of nonlinear least squares to recover the lifetimes and wavelength-dependent fractional Intensities. The latter values determine the emission spectrum and relative intensity of each component in the mixture. Using this technique, we resolved two-component mixtures of fluorescein and 9amlnoacrldlne, 2-(p-toluidinyl)-naphthalene-6-sulfonlc acid and 6-proplonyl-2-(dlmethylamlno)naphthalene, and N-acetyl-L-tyrosinamide and N-acetyl-L-tryptophanamide. In these mixtures the lifetimes differ by about 2-fold. Analysis of simulated data is presented to Illustrate the requirements for a satisfactory resolution. For simulated two-component mixtures, the components can be resolved If the lifetimes differ by 2-fold or greater, even with extensive overlap of the emission spectra. Registry No.

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