Nonparametric Analysis of Time-Resolved Fluorescence Data Based on the Laguerre Expansion Technique

To estimate the intrinsic fluorescence intensity decay of a compound, the excitation light pulse must be deconvolved from the measured fluorescence pulse trace. The most commonly used deconvolution method is the multiexponential least-square iterative reconvolution (LSIR) technique. A variant of LSIR in which the intrinsic fluorescence intensity decay is expressed as an expansion on the discrete time Laguerre basis, was recently introduced. In this study, the performance of the Laguerre deconvolution technique was successfully tested with simulated and fluorescence standard data. It was also demonstrated that the Laguerre deconvolution presents a number of advantages over the classical multiexponential LSIR, including less expensive computational resolution, and the property to generate a unique set of expansion coefficients highly correlated with the intrinsic lifetimes. A novel method for concentration estimation based on the analysis of the Laguerre expansion coefficients was also proposed and successfully applied to different fluorescence standard mixtures, performing even better (error<2%) than more traditional methods of spectral analysis, such as PCR (error<7%) and PLS (error<10%). These findings suggest that the use of Laguerre expansion coefficients represents an alternative nonparametric approach to characterize and discriminate biological systems, in terms of their spectral and lifetime characteristics.