GLOBAL ANALYSIS OF UNMATCHED POLARIZED FLUORESCENCE DECAY CURVES

The performance of the simultaneous analysis of unmatched polarized fluorescence decay curves collected under various experimental conditions, including reference convolution, is investigated systematically for a number of nonassociative (homogeneous) and associative (heterogeneous) model cases with zero limiting anisotropy at long times. The study has been carried out using an improved and rapidly converging algorithm based on the constraint for equal area under the observed and fitted curves. The efficiency of changing experimental conditions such as the orientation of the emission polarizer, the emission/excitation wavelength, and the timing calibration is discussed with respect to the parameter recovery and the power to discriminate between alternative models for the fluorescence anisotropy. When the decay of the total fluorescence and the anisotropy are monoexponential, the combination of decay traces collected at the two extreme orientations of the emission polarizer is sufficient for the parameter recovery when the rotational relaxation time is not too much different from the fluorescence relaxation time and is larger than the timing calibration of the channels. For a single compound system with a multiexponential r(t) the use of different excitation wavelengths is recommended. Different timing calibrations have to be utilized when the relaxation times in the anisotropy decay are widely different. For a mixture of compounds the best results are obtained by using different excitation/emission wavelengths. As an example of the latter a mixture of resorufine and cresyl violet in 1-propanol at 20-degrees-C has been analyzed using reference convolution.