Time-gated Fluorescence Lifetime Imaging in the Near Infrared Regime; A Comprehensive Study Toward In Vivo Imaging

Fluorescence lifetime imaging has an enormous impact on our understanding of biological systems, both in vitro and in vivo. It is a powerful tool for the non-invasive in vitro and in vivo biomolecular and cellular investigations. In particular, it has the potential to target and multiplex different species with high sensitivity and specificity, providing a fast and noninvasive readout at low cost. In this work, we present a time-saving Monte Carlo (MC) simulation of fluorescent photons scattering within a turbid medium, followed by phasor analyzes which enabled the simple multiplexing of different targets in one frame. We then demonstrate a simple and fast method for wide-field FLI in the near-infrared (NIR) region, where tissue scattering and autofluorescence are significantly lower, to enable imaging of deep tissue, using the state-of-the-art timed single-photon avalanche diode array camera (SPAD), SPAD512S. In particular, we show how phasor scattering increases with depth. However, using appropriate background correction, a simple "cut-off" method, and averaging, we can multiplex two targets in one image to a depth of 1 cm in tissue. Our results show that it is possible to perform in vivo FLI under challenging conditions, using standard NIR fluorophores with short lifetimes.