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

This article has been Reviewed by the following groups

Read the full article See related articles

Listed in

Log in to save this article

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.

Article activity feed

  1. 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?