Frequency Domain Multidimensional Fluorescence: A Route to Model-Independent Fluorescence Decay Analysis
Sharon L. Neal · Analytical Chemistry · 1997
A multivariate method for analyzing complex fluorescence decays without model assumptions is described. This method is applicable to the decays of mixtures of spectrally distinct fluorophores. The method does not require specific information concerning the component spectra or lifetimes, but such information can be used when it is available. The decay is analyzed as the complex quantum yield (a function of the steady-state intensity, modulation ratio, and phase angle) measured as a function of modulation frequency and, in most cases, emission wavelength. When the spectra of the mixture components are known, the frequency domain decays of the components can be calculated from complex quantum yield matrices measured at all or selected wavelengths. Matrices describing component photokinetics and relative concentrations are calculated from the frequency domain decays. When the component emission spectra are not known, complex quantum yield matrices can be factored into the emission spectra and frequency domain decays of the sample components via principal components analysis of the matrix. In this paper, the principles of frequency domain decay analysis are discussed. In particular, the variation of the analysis procedure with the amount of data acquired and the extent of the analyst's knowledge of the sample components are described. These procedural variations are illustrated by the analysis of simulated decays and the decays of mixtures of polynuclear aromatic hydrocarbons in isotropic and microheterogeneous solution.