We used frequency-domain fluorometry to resolve multi-exponential decays of fluorescence intensity and anisotropy. The samples are excited with intensity modulated light, at modulation frequencies ranging from 1 to 200 MHz, and we measure the phase-shifts and demodulation factors. The decay times of a three-component mixture were resolved, 1.2, 4.1 and 7.7 nsec, as well as the two anisotropy decay times of the anisotropic rotator perylene. © 1984.

Time-resolved decays of fluorescence anisotropy were obtained from frequency-domain measurements of the phase angle difference between the parallel and perpendicular components of the polarized emission and the ratio of the modulated amplitudes. These data were measured at modulation frequencies ranging from 1 to 200 MHz. To demonstrate the general applicability of this method, we describe the resolution of both simple and complex decays of anisotropy. In particular, we resolved single, double, and triple exponential decays of anisotropy and the hindered rotational motions of fluorophores within lipid bilayers. The ease and rapidity with which these results were obtained indicate that frequency-domain measurements are both practical and reliable for the determination of complex decays of anisotropy.

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.