Rationale: Following an ever-increased focus on personalized medicine, there is a continuing need to develop preclinical molecular imaging modalities to guide the development and optimization of targeted therapies. Near-Infrared (NIR) Macroscopic Fluorescence Lifetime Förster Resonance Energy Transfer (MFLI-FRET) imaging offers a unique method to robustly quantify receptor-ligand engagement in live intact animals, which is critical to assess the delivery efficacy of therapeutics. However, to date, non-invasive imaging approaches that can simultaneously measure cellular drug delivery efficacy and metabolic response are lacking. A major challenge for the implementation of concurrent optical and MFLI-FRET in vivo whole-body preclinical imaging is the spectral crowding and cross-contamination between fluorescent probes. Methods: We report on a strategy that relies on a dark quencher enabling simultaneous assessment of receptor-ligand engagement and tumor metabolism in intact live mice. Several optical imaging approaches, such as in vitro NIR FLI microscopy (FLIM) and in vivo wide-field MFLI, were used to validate a novel donor-dark quencher FRET pair. IRDye 800CW 2-deoxyglucose (2-DG) imaging was multiplexed with MFLI-FRET of NIR-labeled transferrin FRET pair (Tf-AF700/Tf-QC-1) to monitor tumor metabolism and probe uptake in breast tumor xenografts in intact live nude mice. Immunohistochemistry was used to validate in vivo imaging results. Results: First, we establish that IRDye QC-1 (QC-1) is an effective NIR dark acceptor for the FRET-induced quenching of donor Alexa Fluor 700 (AF700). Second, we report on simultaneous in vivo imaging of the metabolic probe 2-DG and MFLI-FRET imaging of Tf-AF700/Tf-QC-1 uptake in tumors. Such multiplexed imaging revealed an inverse relationship between 2-DG uptake and Tf intracellular delivery, suggesting that 2-DG signal may predict the efficacy of intracellular targeted delivery. Conclusions: Overall, our methodology enables for the first time simultaneous non-invasive monitoring of intracellular drug delivery and metabolic response in preclinical studies.

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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.

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