Computational single-photon counting for fast fluorescence lifetime imaging microscopy using single- and multi-photon peak event detection

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Multiphoton and confocal laser scanning fluorescence lifetime imaging microscopy (FLIM) are often limited by slow acquisition due to the dead time of photon-counting and time-tagging analog electronics. Here, we present a solution for faster imaging with lower dead time by directly digitizing the amplified detector output and computationally determining photon counts via GPU-accelerated processing using our custom Single- and multi-photon PEak Event Detection (SPEED) algorithm. The algorithm uses thresholded local maxima detection to temporally localize photon counts in the digitized data, enabling sub-ns dead time between consecutive photon counts.