In vivo quantitative FRET small animal imaging: Intensity versus lifetime-based FRET.
In vivo quantitative FRET small animal imaging: Intensity versus lifetime-based FRET.
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DOI：
10.1016/j.bpr.2023.100110
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发表时间：
2023-06-14
期刊：
影响因子：
--
通讯作者：
Barroso, Margarida
中科院分区：
文献类型：
--
作者：
Smith, Jason T;Sinsuebphon, Nattawut;Rudkouskaya, Alena;Michalet, Xavier;Intes, Xavier;Barroso, Margarida
Förster共振能量转移(FRET)显微镜被用于许多生物物理和生物医学应用，以监测分子间和分子内的相互作用以及2-10纳米范围内的构象变化。FRET目前正被扩展到体内光学成像，其主要应用是使用有机染料或纳米颗粒标记的探针来量化癌症动物模型中药物靶点的参与或药物释放。在这里，我们比较了使用基于强度的FRET(敏化发射FRET分析与使用IVIS成像器的三立方体方法)的FRET定量和使用定制系统使用时间门控增强电荷耦合器件的宏观荧光寿命(Mfli)FRET，用于小动物的活体光学成像。对于这两种方法，都详细描述了量化FRET效率E与FRET中所涉及的给体分子比例乘积所需的分析表达式和实验方案。静脉注射近红外标记的转铁蛋白FRET对，在活体完整裸鼠体内获得转铁蛋白受体-转铁蛋白结合的动态FRET定量，并使用杂交的寡核苷酸在体外以FRET为基准。尽管这两种活体成像技术提供了类似的受体-配体结合的动态趋势，但我们证明了mfli-fret具有显著的优势。虽然使用IVIS成像仪的敏化发射FRET方法需要从三只小鼠获得九个测量结果(其中六个用于校准)，但mfli-fret只需要从一只小鼠收集一个测量结果，尽管在更一般的情况下可能需要一个对照小鼠。因此，基于我们的研究，mfli代表了纵向临床前FRET研究的选择方法，例如在完整的活体小鼠中靶向给药的方法。
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 of the FRET efficiency E and the fraction of donor molecules involved in FRET, , 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.
影响因子：
4.8
作者：
Talati, Ronak;Vanderpoel, Andrew;Eladdadi, Amina;Anderson, Kate;Abe, Ken;Barroso, Margarida
通讯作者：
Barroso, Margarida