| Autor: |
Jason T. Smith, Nattawut Sinsuebphon, Alena Rudkouskaya, Xavier Michalet, Xavier Intes, Margarida Barroso |
| Jazyk: |
angličtina |
| Rok vydání: |
2023 |
| Předmět: |
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| Zdroj: |
Biophysical Reports, Vol 3, Iss 2, Pp 100110- (2023) |
| Druh dokumentu: |
article |
| ISSN: |
2667-0747 |
| DOI: |
10.1016/j.bpr.2023.100110 |
| Popis: |
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 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 three-cube approach using an IVIS imager) and macroscopic fluorescence lifetime (MFLI) FRET using a custom system using a time-gated-intensified charge-coupled device, for small animal optical in vivo imaging. The analytical expressions and experimental protocols required to quantify the product fDE 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 a 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 nine measurements (six 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. |
| Databáze: |
Directory of Open Access Journals |
| Externí odkaz: |
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