We developed an active illumination strategy to acquire optimal fluorescence signals over multi-well plates even in the presence of large fluorophore concentration distributions. We validated the stability of our approach in a 96-well plate setting with fluorophore concentrations ranging >2 orders of magnitude. We report the ability of our method to quantify accurately the lifetime over this concentration range in all wells based on optimized wide-field data and in less than a minute. Our results demonstrate that 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.
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