Biology subjects

Amiet, D.

Publications and source records attributed to Amiet, D..

2 recordsLinked to original sources

bioRxiv2026

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

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.

neuroscience↗

bioRxiv2024

A low-cost FPGA-based approach for pile-up corrected high-speed in vivo FLIM imaging

Intensity-based two-photon microscopy (2PM) is a cornerstone of biomedical research but lacks the ability to measure concentrations, a pivotal task for longitudinal studies and quantitative comparisons. Fluorescence Lifetime Imaging (FLIM) based on Time-Correlated Single Photon Counting (TCSPC) can overcome those limits but suffers from "pile-up" distortions at high photon count rates, severely limiting acquisition speed. We introduce the "laser period blind time" (LPBT) method to correct pile-up distortions in photon counting electronics, enabling reliable low-cost TCSPC-FLIM at high count rates. The correction was implemented on low-cost hardware based on a field programable gate array (FPGA) and validated using a combination of in silico simulations and in vitro, ex vivo and in vivo measurements. The LBPT approach achieves <3% error in lifetime measurements at count rates more than ten times higher than traditional limits, allowing robust FLIM imaging of sub-second metabolite dynamics with subcellular resolution. Our work enables high-precision, cost-effective FLIM imaging at rates comparable to commercial systems and at a fraction of the cost, facilitating the adoption of FLIM across all areas of research needing affordable, quantitative live imaging solutions.

neuroscience↗