Time-resolved fluorescence emission spectra of labeled phospholipid vesicles, as observed using multi-frequency phase-modulation fluorometry

Multi-frequency phase-modulation fluorometry was used to determine the time-resolved spectral parameters of two different fluorescent probes, 6-palmitoyl-2-[(2-trimethylammonium)ethyl[methylamino]naphthalene chloride (Patman) and 2-p-toluidinyl-6-naphthalenesulfonic acid (TNS) in lipid vesicles. The frequency-domain measurements permitted calculation of time-resolved emission spectra, the time-resolved decays of the emission center of gravity and of the emission spectral width. Nanosecond spectral relaxation was found using both probes, and these rates increased with temperature. The detailed time-domain information available using our method indicated that spectral relaxation of TNS is mainly a continuous process, whereas relaxation of Patman shows the characteristic features of a stepwise relaxation. Our results indicate that complex excited-state processes can be quantified using frequency-domain fluorometry. © 1984