Novel Ultra-Fast Deconvolution Method for Fluorescence Lifetime Imaging Microscopy Based on the Laguerre Expansion Technique

A new deconvolution method for fluorescence lifetime imaging microscopy (FLIM) based on the Laguerre expansion technique is presented. The performance of this method was tested on synthetic FLIM images derived from a multiexponential model and from fluorescence lifetime standards, and then compared to standard algorithms of FLIM analysis. Our results demonstrated significant advantages of the Laguerre method over standard algorithms. First, the fluorescence intensity decays of arbitrary form can be estimated at every pixel, without a priori assumption of its functional form. Second, the number of delayed images required to perform deconvolution is relatively low (as low as 5), reducing the acquisition time. Third, ultra-fast light sources are not longer required, making less expensive to perform lifetime imaging. Finally and most important, deconvolution at every pixel is performed in parallel using a common Laguerre basis, thus allowing reducing significantly the computation time (i.e. synthetic 600x600 pixel images can be deconvolved with high accuracy in less than 20 s). Based on these findings, we believe that the Laguerre deconvolution technique represents a more robust and extremely fast analytical method that will allow exploring FLIM in practical real-time applications, such as clinical diagnosis.