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

Abstract

Förster 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 using 3-cube IVIS imager) and macroscopic fluorescence lifetime (MFLI) FRET using a time-resolved ICCD system in small animal opticalin vivoimaging. The analytical expressions and experimental protocols required to quantify FRET efficiency 𝐸 and the fraction of donor molecules actually 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 FRET quantification trends of receptor-ligand engagement, we demonstrate that MFLI FRET has significant advantages. Whereas the sensitized emission FRET approach using IVIS imager required 9 measurements (8 of which are used for calibration) acquired from three mice, MFLI FRET needed only one measurement collected from a single mouse. Hence, MFLI represents the method of choice for FRET measurements in intact, live mice to assess targeted drug delivery in longitudinal preclinical studies, as well as for many otherin vivobiomedical applications.

Probing organoid metabolism using fluorescence lifetime imaging microscopy (FLIM): The next frontier of drug discovery and disease understanding

“…The decay time ('lifetime', tau, t), is an intrinsic characteristic of luminescent molecules, and quantifies the period a molecule remaining in an excited state, between the light absorption and emission events. Importantly, t is affected by the changes in the environment directly surrounding the luminescent molecule, its orientation, rotation, pH, presence of quenchers, temperature and other conditions [25,27]. This can also facilitate the assessment of protein conformation and intramolecular interactions by FLIM, when for example, a luminescent molecule undergoes self-quenching due to the changes in the protein tertiary structure.…”

“…This can also facilitate the assessment of protein conformation and intramolecular interactions by FLIM, when for example, a luminescent molecule undergoes self-quenching due to the changes in the protein tertiary structure. FLIM can also provide quantitative measurements of the Förster Resonance Energy Transfer (FRET) events, in which the energy transfer occurs when a luminescent donor molecule is within 1-10 nm from the acceptor and has correct orientation [27][28][29][30][31]. The flexibility, reliability and adaptability of FLIM makes it a very useful quantitative (and qualitative) modality in numerous biological applications [25].…”

“…Three lifetime range 'domains', often requiring different types of detection equipment can be classified: 0.1~20 ns as 'a conventional FLIM', ~0.5-100 µs as phosphorescence lifetime imaging microscopy (PLIM, mostly for O2 and thermally activated delayed fluorescence temperature imaging) and 100~1000 µs in macro-imaging, phosphorescence quenching microscopy (PQM) and delayed fluorescence-based O2-sensing [32][33][34]. Importantly, FLIM can be assayed in the visible as well as the near infrared range, permitting its application in microscopy as well as in small animal optical imaging [25,27,35,36]. The required lifetime resolution, light source and speed of acquisition dictate different engineering approaches to construct a wide variety of FLIM microscopes.…”