Comparison of NIR FRET pairs for quantitative transferrin-based assay

Sinsuebphon, Nattawut; Bevington, Travis; Zhao, Lingling; Abe, Ken; Barroso, Margarida; Intes, Xavier

doi:10.1117/12.2040097

Cited by 2 publications

(3 citation statements)

References 21 publications

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“…Furthermore, it was found to perform the best for this application compared with five other FRET pairs. 24 Six different acceptor to donor (AD) ratios (0:1, 1:3, 1:2, 1:1, 2:1, and 3:1) were employed to establish FRET parameter estimation accuracy versus temporal data sparsity. The configuration of the multiwell plate and fluorescence intensity (at peak maximum) is provided in Fig.…”

Section: Cell Assay Datamentioning

confidence: 99%

“…These FRET pairs are critical for in vivo applications, as the near-infrared (NIR) spectral window corresponds to the lowest attenuation of photons in biotissues. 22,23 For NIR FRET pairs, the lifetime reduces to a nanosecond or below, with the short-lifetime component associated with the FRETing donor being in the 250-to 350-ps 24 range. For such applications, RLD is not expected to provide accurate results, and biexponential fitting is required.…”

Section: Introductionmentioning

confidence: 99%

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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: Cell Assay Datamentioning

confidence: 99%

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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“…Solutions with various mixture ratios of FRET donor-acceptor pairs were prepared in a 96-well plate and imaged at the specified gate widths. These two dyes were chosen because they provided the best lifetime-based FRET measurements in the NIR range among various other potential FRET pairs [ 36 ]. Both labels were first diluted to a concentration of 50 μg/mL using PBS buffer and then pipetted into the wells in A:D ratios of 0:1, 1:1, 2:1, and 3:1.…”

Section: Methodsmentioning

confidence: 99%

Assessment of Gate Width Size on Lifetime-Based Förster Resonance Energy Transfer Parameter Estimation

Chen

Intes

2015

Photonics

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Förster Resonance Energy Transfer (FRET) enables the observation of interactions at the nanoscale level through the use of fluorescence optical imaging techniques. In FRET, fluorescence lifetime imaging can be used to quantify the fluorescence lifetime changes of the donor molecule, which are associated with proximity between acceptor and donor molecules. Among the FRET parameters derived from fluorescence lifetime imaging, the percentage of donor that interacts with the acceptor (in proximity) can be estimated via model-based fitting. However, estimation of the lifetime parameters can be affected by the acquisition parameters such as the temporal characteristics of the imaging system. Herein, we investigate the effect of various gate widths on the accuracy of estimation of FRET parameters with focus on the near-infrared spectral window. Experiments were performed in silico, in vitro, and in vivo with gate width sizes ranging from 300 ps to 1000 ps in intervals of 100 ps. For all cases, the FRET parameters were retrieved accurately and the imaging acquisition time was decreased three-fold. These results indicate that increasing the gate width up to 1000 ps still allows for accurate quantification of FRET interactions even in the case of short lifetimes such as those encountered with near-infrared FRET pairs.

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Comparison of NIR FRET pairs for quantitative transferrin-based assay

Cited by 2 publications

References 21 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

Assessment of Gate Width Size on Lifetime-Based Förster Resonance Energy Transfer Parameter Estimation

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