Abstract
Wide-field fluorescence lifetime imaging allows for fast imaging of large sample areas at the cost of low sensitivity to weak fluorescence signals. To overcome this challenge, we developed an active wide-field illumination (AWFI) strategy to optimize the impinging spatial intensity for acquiring optimal fluorescence signals over the whole sample. We demonstrated the ability of AWFI to accurately estimate lifetimes from a multiwell plate sample with concentrations ranging over two orders of magnitude. We further reported its successful application to a quantitative Förster resonance energy transfer lifetime cell-based assay. Overall, this method allows for enhanced accuracy in lifetime-based imaging at high acquisition speed over samples with large fluorescence intensity distributions.