FLIM-FRET for Cancer Applications

Rajoria, Shilpi; Zhao, Lingling; Intes, Xavier; Barroso, Margarida

doi:10.2174/2211555203666141117221111

Cited by 64 publications

(59 citation statements)

References 111 publications

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“…However, FRET based on FLIM is still mainly confined to microscopy applications with visible fluorophore FRET pairs and long acquisition times. In order to translate FRET assays to highthroughput in vitro and in vivo applications for drug discovery, 15,16 it is critical to identify strategies that will decrease measurement acquisition times without compromising quantitative FLIM-FRET analysis.…”

Section: Introductionmentioning

confidence: 99%

Reduced temporal sampling effect on accuracy of time-domain fluorescence lifetime Förster resonance energy transfer

et al. 2014

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Abstract. Fluorescence lifetime imaging (FLIM) aims at quantifying the exponential decay rate of fluorophores to yield lifetime maps over the imaged sample. When combined with Förster resonance energy transfer (FRET), the technique can be used to indirectly sense interactions at the nanoscale such as protein-protein interactions, protein-DNA interactions, and protein conformational changes. In the case of FLIM-FRET, the fluorescence intensity decays are fitted to a biexponential model in order to estimate the lifetime and fractional amplitude coefficients of each component of the population of the donor fluorophore (quenched and nonquenched). Numerous time data points, also called temporal or time gates, are typically employed for accurately estimating the model parameters, leading to lengthy acquisition times and significant computational demands. This work investigates the effect of the number and location of time gates on model parameter estimation accuracy. A detailed model of a FLIM-FRET imaging system is used for the investigation, and the simulation outcomes are validated with in vitro and in vivo experimental data. In all cases investigated, it is found that 10 equally spaced time gates allow robust estimation of model-based parameters with accuracy similar to that of full temporal datasets (90 gates).

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Section: Introductionmentioning

confidence: 99%

Reduced temporal sampling effect on accuracy of time-domain fluorescence lifetime Förster resonance energy transfer

et al. 2014

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“…Historically, FRET has been sensed via intensity-based approaches, but the current FRET quantification standard relies on fluorescence lifetime imaging microscopy (FLIM). FLIM has the main benefit of a robust and highly reproducible quantification of FRET with less experimentally cumbersome protocols [9,10]. However, translation of FLIM-FRET to macroscale FRET applications was limited until the development of MFLI platforms and redshifting of the FRET fluorophore pair [11,12].…”

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confidence: 99%

Comparison of illumination geometry for lifetime‐based measurements in whole‐body preclinical imaging

Sinsuebphon

Rudkouskaya

Barroso

et al. 2018

Journal of Biophotonics

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Macroscopic fluorescence lifetime imaging (MFLI) has been proved to be an accurate tool to quantify Förster resonance energy transfer (FRET) lifetime-based assessment of receptor-ligand engagement in vitro and in vivo. Herein, we report on the quantitative comparison of MFLI for whole-body preclinical studies in transmittance and reflectance geometries. The comparative study was conducted for both in vitro and in vivo conditions. FRET quantification performance in both geometries was similar in high fluorescence concentration samples. However, the reflectance geometry performed better at low fluorescence concentration. In addition, reflectance geometry could acquire subsurface imaging of the main whole-body organs of small animals without being compromised by tissue attenuation.

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“…Upon donor excitation, FLI estimates FRET occurrence by determining the reduction of the fluorescence lifetime of the donor molecule, when in nanometer (2-10nm) proximity of an acceptor molecule. [12][13][14] When applied to receptor-ligand systems, FRET occurs when donor-labeled and acceptor-labeled ligands/antibodies bind to dimerized or cross-linked receptors. [15][16][17][18][19][20][21] Hence, FLI FRET acts as a direct reporter of receptor engagement and internalization via the measurement of the fraction of labeled-donor entity undergoing binding to its respective receptor and subsequent internalization.…”

Section: Introductionmentioning

confidence: 99%

Multiplexed Non-invasive in vivo Imaging to Assess Metabolism and Receptor Engagement in Tumor Xenografts

Rudkouskaya

Sinsuebphon

Ochoa

et al. 2019

Preprint

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Following an ever-increased focus on personalized medicine, there is a continuing need to develop preclinical molecular imaging modalities to guide the development and optimization of targeted therapies. To date, non-invasive quantitative imaging modalities that can comprehensively assess simultaneous cellular drug delivery efficacy and therapeutic response are lacking. In this regard, Near-Infrared (NIR) Macroscopic Fluorescence Lifetime Förster Resonance Energy Transfer (MFLI-FRET) imaging offers a unique method to robustly quantify receptor-ligand engagement in vivo and subsequent intracellular internalization, which is critical to assess the delivery efficacy of targeted therapeutics. However, implementation of multiplexing optical imaging with FRET in vivo is challenging to achieve due to spectral crowding and cross-contamination. Herein, we report on a strategy that relies on a dark quencher that enables simultaneous assessment of receptor-ligand engagement and tumor metabolism in intact live mice. First, we establish that IRDye QC-1 (QC-1) is an effective NIR dark acceptor for the FRET-induced quenching of donor Alexa Fluor 700 (AF700) using in vitro NIR FLI microscopy and in vivo wide-field MFLI imaging. Second, we report on simultaneous in vivo imaging of the metabolic probe IRDye 800CW 2-deoxyglucose (2-DG) and MFLI-FRET imaging of NIR-labeled transferrin FRET pair (Tf-AF700/Tf-QC-1) uptake in tumors. Such multiplexed imaging revealed an inverse relationship between 2-DG uptake and Tf intracellular delivery, suggesting that 2-DG signal may predict the efficacy of intracellular targeted delivery. Overall, our methodology enables for the first time simultaneous non-invasive monitoring of intracellular drug delivery and metabolic response in preclinical studies.

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FLIM-FRET for Cancer Applications

Cited by 64 publications

References 111 publications

Reduced temporal sampling effect on accuracy of time-domain fluorescence lifetime Förster resonance energy transfer

Reduced temporal sampling effect on accuracy of time-domain fluorescence lifetime Förster resonance energy transfer

Comparison of illumination geometry for lifetime‐based measurements in whole‐body preclinical imaging

Multiplexed Non-invasive in vivo Imaging to Assess Metabolism and Receptor Engagement in Tumor Xenografts

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