Resolution of multicomponent fluorescence emission using frequency-dependent phase angle and modulation spectra

We describe a new fluorescence method that allows the resolution of both the decay times and emission spectra of mixtures of fluorophores. This method is completely general and does not require any assumptions or knowledge of the decay times or emission spectra of the individual fluorophores. We use the phase angle spectra and modulation spectra of the mixture, measured over a range of suitable light modulation frequencies and emission wavelengths. These data are analyzed by nonlinear least-squares analysis to recover the emission spectra and the associated decay times. The principle of the method and the nature of the data are illustrated by using two-component mixtures with increasing spectral overlap. We then demonstrate the recovery of minor components, of structure emission spectra, and of a three-component mixture with completed overlapping emission spectra. And finally, we describe the resolution of a two-component mixture with decay times of 0.8 and 1.4 ns using modulation frequencies up to 774 MHz.