Time-gated Fluorescence Lifetime Imaging in the Near Infrared Regime

Fluorescence lifetime (FLT) provides a concentration-independent contrast mechanism that is highly sensitive to the local biochemical environment, making it a powerful tool for biomedical optical imaging. In this work, we present a time-gated near-infrared (NIR) fluorescence lifetime imaging system operating in the phasor domain, based on a single-photon avalanche diode (SPAD) camera. The system architecture, optical configuration, and gating strategy are described, together with a MATLAB-based lifetime extraction algorithm optimized for accurate FLT recovery under low-photon and high-background conditions. Reliable lifetime measurements were ensured through careful impulse response function (IRF) calibration and deconvolution. Phasor-based analysis enabled clear discrimination of dyes exhibiting distinct fluorescence lifetimes, including nanoparticle-conjugated dyes. To evaluate performance under in vivo–relevant conditions, tissue-mimicking phantoms with skin-like optical properties in the near-infrared region of the electromagnetic spectrum and thicknesses of 0.1–1 cm were placed above the dye samples. The results demonstrate robust lifetime differentiation in the phasor domain, highlighting the potential of this time-gated SPAD-based approach for future real-time and non-invasive fluorescence lifetime imaging applications.