Abstract
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.
Competing Interest Statement
The authors have declared no competing interest.
