PURELIGHT: a quantitative photon-counting framework unifying intensity and lifetime imaging at video rate across detector technologies

Quantitative fluorescence microscopy demands photon efficiency, speed, and precise intensity and lifetime measurements. Time-correlated single-photon counting (TCSPC) measures both intensity and lifetime concurrently, but photon pile-up distorts both signals at high count rates, precluding rapid acquisitions. Current corrections eliminate photons, compromise intensity, or necessitate specialized detectors.

In this article, we present PURELIGHT, a unified hardware and software platform that concurrently recovers accurate intensities and lifetimes even at count rates surpassing traditional pile-up boundaries. PURELIGHT functions with hybrid photodetectors, silicon photomultipliers, and photomultiplier tubes while retaining over three times more photons compared to alternative methods.

Utilizing two-photon imaging, we illustrate PURELIGHT's enhanced accuracy and spatial contrast, including video-rate subcellular lifetime imaging in conscious mice, an unprecedented ratiometric modality, and crosstalk-free temporal multiplexing. By eliminating the constraints that have restricted TCSPC to low-signal applications, PURELIGHT encourages the widespread use of quantitative, photon-efficient microscopy in various life sciences disciplines.