PURELIGHT: a quantitative photon-counting framework unifying intensity and lifetime imaging at video rate across detector technologies
- 1Institute of Pharmacology and Toxicology, University of Zurich, Zurich, Switzerland
Abstract
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.
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BibTeX
@misc{moros2026purelight,
title = {{PURELIGHT: a quantitative photon-counting framework unifying intensity and lifetime imaging at video rate across detector technologies}},
author = {F. Velasquez Moros and D. Amiet and D. Skwarzynska and R. M. Meister and U. Dalvi and S. Ma and P. Rupprecht and S. Han and P. Bethge and L. Krainer and G. Acconcia and I. Rech and F. Helmchen and P. Zbinden and A. S. Saab and B. Weber and L. Ravotto},
howpublished = {bioRxiv},
year = {2026},
month = aug,
doi = {10.64898/2026.08.28.747260},
url = {https://www.biorxiv.org/content/10.64898/2026.08.28.747260v1},
note = {CC BY},
}
RIS
TY - UNPB
TI - PURELIGHT: a quantitative photon-counting framework unifying intensity and lifetime imaging at video rate across detector technologies
AU - F. Velasquez Moros
AU - D. Amiet
AU - D. Skwarzynska
AU - R. M. Meister
AU - U. Dalvi
AU - S. Ma
AU - P. Rupprecht
AU - S. Han
AU - P. Bethge
AU - L. Krainer
AU - G. Acconcia
AU - I. Rech
AU - F. Helmchen
AU - P. Zbinden
AU - A. S. Saab
AU - B. Weber
AU - L. Ravotto
PY - 2026
DA - 2026/08/28
PB - bioRxiv
DO - 10.64898/2026.08.28.747260
UR - https://www.biorxiv.org/content/10.64898/2026.08.28.747260v1
AB - 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.
ER -