GLOBAL ANALYSIS OF STEADY-STATE POLARIZED FLUORESCENCE-SPECTRA USING TRILINEAR CURVE RESOLUTION

Global analysis using trilinear curve resolution is described and show n to be a powerful method for the resolution of polarized fluorescence data arrays, in which the measured fluorescence intensity is a separa ble function of polarization orientation, excitation wavelength, and e mission wavelength. This methodology is applicable to mixtures the com ponents of which have linearly independent excitation and emission spe ctra and distinct anisotropies. Normalized excitation and emission spe ctra of individual components can be uniquely determined without prior assumptions concerning spectral shapes (e.g., sum of Gaussians) and w ithout the uncertainties inherent in bilinear techniques such as princ ipal component analysis or factor analysis. The normalized excitation and emission vectors are combined with the total absorption spectrum o f the multicomponent mixture to compute absolute absorption and emissi on spectra. The precision of this methodology is evaluated as a functi on of noise, overlap, relative intensity, and anisotropy difference be tween components using simulated mixtures of the DNA bases. The abilit y of this method to extract individual spectra from steady-state fluor escence data arrays is illustrated for mixtures containing two and thr ee components.