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Active illumination for wide-field time-resolved fluorescence imaging

The large dynamic range of fluorescence emission collected is one of the major challenges in wide-field fluorescence lifetime imaging. To overcome this challenge, we developed an active illumination strategy to acquire optimal fluorescence signals over the sample imaged even in the presence of large fluorophore concentration distributions. We validated the stability 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 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.