Global analysis of steady-state polarized fluorescence spectra using trilinear curve resolution

Global analysis using trilinear curve resolution is described and shown to be a powerful method for the resolution of polarized fluorescence data arrays, in which the measured fluorescence intensity is a separable function of polarization orientation, excitation wavelength, and emission wavelength. This methodology is applicable to mixtures the components of which have linearly independent excitation and emission spectra and distinct anisotropies. Normalized excitation and emission spectra of individual components can be uniquely determined without prior assumptions concerning spectral shapes (e.g., sum of Gaussians) and without the uncertainties inherent in bilinear techniques such as principal component analysis or factor analysis. The normalized excitation and emission vectors are combined with the total absorption spectrum of the multicomponent mixture to compute absolute absorption and emission spectra. The precision of this methodology is evaluated as a function of noise, overlap, relative intensity, and anisotropy difference between components using simulated mixtures of the DNA bases. The ability of this method to extract individual spectra from steady-state fluorescence data arrays is illustrated for mixtures containing two and three components.