Quantitative fluorescence microscopy requires photon-efficiency, speed and accurate intensity and lifetime measurements. Time-correlated single-photon counting (TCSPC) simultaneously captures intensity and lifetime, but photon pile-up distorts both signals at high count rates, preventing fast acquisitions. Existing corrections discard photons, distort intensity, or require specialized detectors. Here we introduce PURELIGHT, an integrated hardware and software framework that simultaneously recovers undistorted intensities and lifetimes at count rates far beyond conventional pile-up limits. PURELIGHT works with hybrid photodetectors, silicon photomultipliers and photomultiplier tubes while retaining over three times more photons than alternative approaches. Using two-photon imaging, we showcase PURELIGHT's superior accuracy and spatial contrast, demonstrating video-rate subcellular lifetime imaging in awake mice, a unique lifetime-calibrated ratiometric modality and crosstalk-free temporal multiplexing. By removing the limits that have confined TCSPC to low-signal applications, PURELIGHT promotes the adoption of quantitative, photon-efficient microscopy across the life sciences.
Velasquez Moros, F., Amiet, D., Skwarzynska, D., Meister, R. M., Dalvi, U., Ma, S., Rupprecht, P., Han, S., Bethge, P., Krainer, L., Acconcia, G., Rech, I., Helmchen, F., Zbinden, P., Saab, A. S., Weber, B., Ravotto, L.
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