in vivoquantitative FRET small animal imaging: intensity versus lifetime-based FRET

Abstract

ABSTRACTFörster Resonance Energy Transfer (FRET) microscopy is used in numerous biophysical and biomedical applications to monitor inter- and intramolecular interactions and conformational changes in the 2–10 nm range. FRET is currently being extended toin vivooptical imaging, its main application being in quantifying drug-target engagement or drug release in animal models of cancer using organic dye or nanoparticle-labeled probes. Herein, we compared FRET quantification using intensity-based FRET (sensitized emission FRET analysis with the 3-cube approach using an IVIS imager) and macroscopic fluorescence lifetime (MFLI) FRET using a custom system using a time-gated ICCD, for small animal opticalin vivoimaging. The analytical expressions and experimental protocols required to quantify the productfDEof the FRET efficiencyEand the fraction of donor molecules involved in FRET,fD, are described in detail for both methodologies. Dynamicin vivoFRET quantification of transferrin receptor-transferrin binding was acquired in live intact nude mice upon intravenous injection of near infrared-labeled transferrin FRET pair and benchmarked againstin vitroFRET using hybridized oligonucleotides. Even though bothin vivoimaging techniques provided similar dynamic trends for receptor-ligand engagement, we demonstrate that MFLI FRET has significant advantages. Whereas the sensitized emission FRET approach using the IVIS imager required 9 measurements (6 of which are used for calibration) acquired from three mice, MFLI FRET needed only one measurement collected from a single mouse, although a control mouse might be needed in a more general situation. Based on our study, MFLI therefore represents the method of choice for longitudinal preclinical FRET studies such as that of targeted drug delivery in intact, live mice.WHY IT MATTERSFRET measurements in live animals open a unique window into drug-target interaction monitoring, by sensing the close proximity between a donor and acceptor-labeled molecular probes. To perform these measurements, a 3-cube fluorescent intensity measurement strategy can be adopted, as is common forin vitroFRET microscopy studies. However, it is challenging to translate this already cumbersome approach toin vivosmall animal imaging. Here, we compare this standard approach, for which we provide a revised analytical framework, to a conceptually much simpler and more powerful one based on fluorescence lifetime measurements. Our results demonstrate that the technical challenge ofin vivofluorescence lifetime macroscopic imaging is well worth surmounting to obtain quantitative, whole-animal information regarding molecular drug-target engagement.