Up to 200% of the excitation frequency in compressive FLIM: a constraint-free pile-up correction methodology for advanced biomedical imaging

Time-correlated single-photon counting (TCSPC) stands at the forefront of low-intensity light detection techniques, providing unparalleled sensitivity and timing precision in the reconstruction of faint and rapid optical signals. The growing demand for faster data acquisition in advanced applications has driven considerable interest in developing new strategies to overcome its historical speed limitation. Typically, the count rate of a TCSPC acquisition chain is restricted to just a small fraction (1-5%) of the laser excitation frequency to prevent the onset of <i>pile-up</i> distortion. In this work, we exploit the capabilities of a novel distortion correction methodology that does not require any constraints on detector dead time, illumination intensity, or prior knowledge of the signal shape. We show its performance in a fluorescence lifetime imaging microscopy (FLIM) application, by leveraging a custom-designed detection module based on a silicon photomultiplier (SiPM) as a single-pixel camera (SPC). By applying the correction methodology to each acquired histogram prior to reconstructing the SPC image for each frame, we demonstrate that sequential acquisitions can achieve rates of up to 20 frames per second, with a photon count rate achieving for the first time 200% of the laser excitation frequency. This result is accomplished by introducing minimal distortion effects in the acquired optical signals, ensuring both the integrity and accuracy of the fluorescence lifetime measurements.