Deconvolution in Fluorescence Lifetime imaging microscopy (FLIM)

Abstract

Fluorescence lifetime imaging microscopy (FLIM) is an important technique to understand the chemical microenvironment in cells and tissues since it provides additional contrast compared to conventional fluorescence imaging. When two fluorophores within a diffraction limit are excited, the resulting emission leads to nonlinear spatial distortion and localization effects in intensity (magnitude) and lifetime (phase) components. To address this issue, in this work, we provide a theoretical model for convolution in FLIM to describe how the resulting behavior differs from conventional fluorescence microscopy. We then present a Richardson-Lucy (RL) based deconvolution including total variation (TV) regularization method to correct for the distortions in FLIM measurements due to optical convolution, and experimentally demonstrate this FLIM deconvolution method on a multi-photon microscopy (MPM)-FLIM images of fluorescent-labeled fixed bovine pulmonary arterial endothelial (BPAE) cells.