Quantifying Lifetime Uncertainty in Gated-ICCD Fluorescence Lifetime Imaging

<p dir="ltr">Fluorescence lifetime imaging (FLI) is a versatile technique for quantifying molecular processes in biomedical applications. Wide-field FLI analyzes fluorescence temporal decays computationally, often using intensified charge-coupled device (ICCD) cameras for both microscopic (FLIM) and macroscopic (MFLI) imaging. While uncertainty in lifetime parameter estimation has been extensively studied for photon-counting detectors, ICCDs pose unique challenges due to their nonlinear noise characteristics. Factors such as exposure time, mi- crochannel plate (MCP) gain, and illumination power significantly influence recorded fluorescence intensities. These variations complicate accurate photon count es- timation and, consequently, the determination of un- certainty in decay parameters. To address these chal- lenges, we propose a heuristic method for estimating un- certainties in ICCD-based fluorescence lifetimes. This method directly correlates uncertainty with recorded intensity and camera settings. It replaces the conven- tional shot-noise-limited uncertainty model used for photon-counting detectors with a modified expression, supported by a lookup table (LUT) that accounts for spe- cific experimental setups and camera configurations. We validated this approach through extensive studies with phantoms and demonstrated its in vivo application by imaging HER2+ breast tumor xenografts in nude mice. The imaging utilized Trastuzumab labeled with Alexa Fluor 700 and IRDye 800 2DG, highlighting the method’s reliability and effectiveness in preclinical biomedical imaging scenarios.</p>