in vivo quantitative FRET small animal imaging: intensity versus lifetime-based FRET

Abstract

Forster 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 to in vivo optical 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 optical in vivo imaging. The analytical expressions and experimental protocols required to quantify the product fD E of the FRET efficiency E and the fraction of donor molecules involved in FRET, fD, are described in detail for both methodologies. Dynamic in vivo FRET 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 against in vitro FRET using hybridized oligonucleotides. Even though both in vivo imaging 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 for in vitro FRET microscopy studies. However, it is challenging to translate this already cumbersome approach to in vivo small 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 of in vivo fluorescence lifetime macroscopic imaging is well worth surmounting to obtain quantitative, whole-animal information regarding molecular drug-target engagement.