Enhanced Dynamic Range and Accuracy of Fluorescence Lifetime Imaging by Active Illumination

Wide-field fluorescence lifetime imaging allows for fast imaging of large sample at the cost of limited sensitivity to weak fluorescence signals. To overcome this limitation, we develop an active illumination strategy that optimizes the spatial parameters of the illumination in order to acquire optimal wide-field fluorescence signals even in the presence of large fluorophore concentration distributions. We validate the sensitivity gain of our approach in a multi-well plate setting with fluorophore concentrations ranging >2 orders of magnitude. We report the ability of our method to retrieve accurately the lifetime over this concentration range based on optimized wide-field data. Our results demonstrate that an active wide-field illumination can improve the signal-to-noise ratio and weak-signal sensitivity for enhanced accuracy of fluorescence decay curve fitting and lifetime estimation at high acquisition speed.