Toward Real-Time Tcspc-Flim: Unconstrained Pile-Up Correction Method for Distortion-Free Measurements at Extreme Count Rates

Fluorescence Lifetime Imaging Microscopy (FLIM) is a powerful tool for investigating biological and physiological processes down to the subcellular level, by leveraging the temporal properties of weak fluorescence signals. Among photon detection techniques, Time-Correlated Single-Photon Counting (TCSPC) is the gold standard for achieving high temporal resolution and sensitivity. However, traditional TCSPC systems historically suffer from slow acquisition speed, especially for large-field imaging. Wide-field acquisition using parallel detector arrays increases hardware complexity, while single-pixel detectors offer a simpler alternative but rely on spatial scanning, which prolongs acquisition times. In this context, Single-Pixel Cameras (SPCs) combined with Compressive Sensing (CS) have emerged as a cost-effective solution, significantly reducing the number of required measurements. However, acquisition speed in SPC-FLIM is fundamentally limited by pile-up effects inherent to conventional TCSPC systems, which restrict photon count rates to below 5% of the laser repetition frequency. In this work, we demonstrate that integrating a custom ultra-low dead time detection module as SPC into a wide-field FLIM system, along with a novel pile-up correction algorithm, enables video-rate imaging at 20 frames per second achieving photon count rates exceeding 200% of the excitation frequency, with minimal fluorescence lifetime estimation error.