A SYSTEMATIC STUDY OF THE GLOBAL ANALYSIS OF MULTIEXPONENTIAL FLUORESCENCE DECAY SURFACES USING REFERENCE CONVOLUTION

The performance of the simultaneous (global) analysis of multiexponential fluorescence decay surfaces using reference convolution is investigated in a systematic way using synthetic and experimental data sets. It is shown that the increased model discrimination ability and the more accurate parameter recovery by global analysis as compared to single-curve analysis originate from combining decay traces with differing contributions of the decay components. Simultaneous analysis of decay traces in which the contributions of the components are changed as much as possible is the most beneficial. For decay surfaces collected as a function of the emission (excitation) wavelength, this implies that decays with minimal overlap between the emission (absorption) spectra associated with the decay components will contribute the most to the improved model discrimination and parameter recovery. Since this overlap will often be small at the edges of the emission (absorption) spectrum, decays collected there should be included in the global analysis to enhance its performance. For fluorescence decays with widely differing decay rates, one can change the actual contributions of the various kinetic components by collecting decay traces at multiple timing calibrations. Analyzing the resulting decay surface improves the accuracy and precision of the recovery of a short-lived component with a minor contribution. When the reference lifetime is a variable parameter in the analysis, it is advantageous to simultaneously analyze with different references, especially when a reference lifetime is simular to one of the sample decay times. It is further demonstrated that including more decay traces in the global decay surface does not necessarily imply a better model distinction capability. For a fixed number of total counts and a fixed time window in which the fluorescence decay surface is observed, the ability to resolve closely spaced lifetimes increases as the number of channels is reduced. This is due to the enhanced signal-to-noise ratio of the individual decay curves. © 1990 American Chemical Society.