A low-cost FPGA-based approach for pile-up corrected high-speed in vivo FLIM imaging
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Abstract
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.
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Despite these advantages, the widespread adoption of FLIM has been constrained by the lack of straightforward approaches to correct pile-up distortions that would otherwise limit its application for capturing biological dynamics on second and sub-second timescales.
I found your work on LPBT correction for pile-up distortions very impressive. I'm curious if you could propose a straightforward experiment to determine whether a commercial FLIM system is significantly affected by pile-up distortions? Perhaps using your fluorescein with potassium iodide approach as a standardized test?
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