TRANES Spectra of Fluorescence Probes in Lipid Bilayer Membranes: An Assessment of Population Heterogeneity and Dynamics

Time-resolved fluorescence of eight fluorescence probes were studied in EggPC bilayer membrane vesicles. Emission wavelength dependent fluorescence decays were analyzed in a model-independent way to obtain time resolved area normalized emission spectra (TRANES). The TRANES spectra of the probes studied were classified into four types: (1) spectra that are identical at all time (one emissive species), (2) spectra that show an isoemissive point (two emissive species), (3) spectra that shift continuously with time (slow solvation dynamics or multiple species), and (4) spectra that shift for a short time and thereafter one or two emissive species are indicated. The TRANES spectra of these eight probes, except RH421, belong to the type 1, 2, or 4. The continuous shift of the TRANES spectra that was observed for the probe RH421 is attributed to multiple ground state species and not due to slow solvation dynamics.