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

Significance: Intensity-based two-photon microscopy is a cornerstone of neuroscience 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. Aim: 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. Approach: Using a realistic simulation of the TCSPC data collection, we evaluated the LPBT method's performance . The correction was then implemented on low-cost hardware based on a field programable gate array and validated using , , and measurements. Results: The LBPT approach achieves error in lifetime measurements at count rates more than 10 times higher than traditional limits, allowing robust FLIM imaging of subsecond metabolite dynamics with subcellular resolution. Conclusions: We enable high-precision, cost-effective FLIM imaging at acquisition speeds comparable with state-of-the-art commercial systems, facilitating the adoption of FLIM in neuroscience and other fields of research needing robust quantitative live imaging solutions.