The ability to probe deep tissues with light for biomedical applications has been recognized since the scientific reports published as early as the late 1800s for monitoring brain hemorrhage¹ and early 1900s for imaging breast cancer² and performing tissue oximetry.^3,4 Since then, diffuse optical imaging techniques have greatly benefited numerous biomedical fields. Mainly, the goal of these optical techniques is to characterize optical properties of tissue using light from the ultraviolet to the infrared spectral region. The specific light–tissue interaction enables monitoring of a vast array of tissue characteristics, including structural, physiological, metabolic, and molecular properties.

Förster Resonance Energy Transfer (FRET) enables the observation of interactions at the nanoscale level through the use of fluorescence optical imaging techniques. In FRET, fluorescence lifetime imaging can be used to quantify the fluorescence lifetime changes of the donor molecule, which are associated with proximity between acceptor and donor molecules. Among the FRET parameters derived from fluorescence lifetime imaging, the percentage of donor that interacts with the acceptor (in proximity) can be estimated via model-based fitting. However, estimation of the lifetime parameters can be affected by the acquisition parameters such as the temporal characteristics of the imaging system. Herein, we investigate the effect of various gate widths on the accuracy of estimation of FRET parameters with focus on the near-infrared spectral window. Experiments were performed in silico, in vitro, and in vivo with gate width sizes ranging from 300 ps to 1000 ps in intervals of 100 ps. For all cases, the FRET parameters were retrieved accurately and the imaging acquisition time was decreased three-fold. These results indicate that increasing the gate width up to 1000 ps still allows for accurate quantification of FRET interactions even in the case of short lifetimes such as those encountered with near-infrared FRET pairs.

1 We introduce a simple new approach for time-resolved multiplexed analysis of complex systems using near-infrared (NIR) dyes, applicable to in vitro and in vivo studies. We first show that fast and precise in vitro quantification of NIR fluorophores lifetime and stoichiometry can be done using phasor analysis, a computationally efficient and user-friendly representation of complex fluorescence intensity decays obtained with pulsed laser excitation. We apply this approach to the study of binding equilibria by Förster resonant energy transfer (FRET), using two different model systems: primary/secondary antibody binding in vitro and ligand/receptor binding in cell cultures. We then extend our demonstration to dynamic imaging of the pharmacokinetics of transferrin binding to the transferrin receptor in live mice, elucidating the kinetic of differential transferrin accumulation in specific organs, straightforwardly differentiating specific from non-specific binding. Our method, implemented in a freely-available software package, has all the advantages of time-resolved NIR imaging, including better tissue penetration and background-free imaging, but simplifies and considerably speeds up data processing and interpretation, while remaining quantitative. These advances make this method attractive and of broad applicability for in vitro and in vivo molecular imaging, and could be extended to applications as diverse as image guided-surgery or optical tomography.

Lifetime-based FRET was used to compare the intracellular delivery of PEGylated and non-PEGylated probes in vitro and in vivo for applications in transferrin-based drug delivery.