Fluorophore saturation is the key factor limiting the speed and excitation range of fluorescence lifetime imaging microscopy (FLIM). For example, fluorophore saturation causes incorrect lifetime measurements when using conventional frequency-domain FLIM at high excitation powers. In this Letter, we present an analytical theoretical description of this error and present a method for compensating for this error in order to extract correct lifetime measurements in the limit of fluorophore saturation. We perform a series of simulations and experiments to validate our methods. The simulations and experiments show a 13.2× and a 2.6× increase in excitation range, respectively. The presented method is based on algorithms that can be easily applied to existing FLIM setups.
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