Calibration Approach for Fluorescence Lifetime Determination for Applications Using Time-Gated Detection and Finite Pulse Width Excitation

Keller, Scott B.; Dudley, Jonathan; Binzel, Katherine; Jasensky, Joshua; Pedro, Hector Michael de; Frey, Eric W.; Urayama, Paul

doi:10.1021/ac801252q

Cited by 4 publications

(4 citation statements)

References 23 publications

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“…A major drawback, however, is that RLD assumes the IRF of the system to be negligible. 19 This assumption can hold for some visible FRET pairs that exhibit lifetimes of a few nanoseconds compared with the typical ∼250-to 300-ps widths of experimental IRFs. However, this is not the case when using FRET pairs that are red shifted.…”

Section: Introductionmentioning

confidence: 98%

“…The most common approach, which takes into account the temporal effect of the imaging system characteristics, consists of fitting the measurement to an exponential-decay model convolved with an experimental IRF using a least-squares (LSQR) method. 19 An alternate method to exponential fitting is rapid lifetime determination (RLD). Originally developed for single-exponential decays, 20 RLD has been expanded and applied to double-exponential decays 21 for applications such as FRET.…”

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: Introductionmentioning

confidence: 98%

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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“…To retrieve the lifetime and fractional abundance of the quenched donor population, the asymptotic portions of the TPSF were fit with the bi-exponential model. Techniques such as rapid lifetime determination (RLD) are commonly employed with visible fluorophores, but they are not accurate when the IRF temporal width is not negligible compared to the considered lifetime [ 35 ]. In the NIR, the IRF temporal width is on the same order as the quenched donor lifetime or even larger.…”

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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“…These comprehensive temporal data sets result in accurate parameter estimates, but at the cost of increased imaging time, limiting the applicability to relatively few in vivo or high-throughput applications. Recently, methods such as rapid lifetime determination (RLD) [ 13 , 14 ] and phasor analysis [ 15 , 16 ] have gained popularity as they circumvent the need for iterative fitting based on large temporal data sets. These non-fitting methods directly calculate parameters of interest such as fluorescence lifetime and FRET fractions.…”

Section: Introductionmentioning

confidence: 99%

Temporal Data Set Reduction Based on D-Optimality for Quantitative FLIM-FRET Imaging

2015

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Fluorescence lifetime imaging (FLIM) when paired with Förster resonance energy transfer (FLIM-FRET) enables the monitoring of nanoscale interactions in living biological samples. FLIM-FRET model-based estimation methods allow the quantitative retrieval of parameters such as the quenched (interacting) and unquenched (non-interacting) fractional populations of the donor fluorophore and/or the distance of the interactions. The quantitative accuracy of such model-based approaches is dependent on multiple factors such as signal-to-noise ratio and number of temporal points acquired when sampling the fluorescence decays. For high-throughput or in vivo applications of FLIM-FRET, it is desirable to acquire a limited number of temporal points for fast acquisition times. Yet, it is critical to acquire temporal data sets with sufficient information content to allow for accurate FLIM-FRET parameter estimation. Herein, an optimal experimental design approach based upon sensitivity analysis is presented in order to identify the time points that provide the best quantitative estimates of the parameters for a determined number of temporal sampling points. More specifically, the D-optimality criterion is employed to identify, within a sparse temporal data set, the set of time points leading to optimal estimations of the quenched fractional population of the donor fluorophore. Overall, a reduced set of 10 time points (compared to a typical complete set of 90 time points) was identified to have minimal impact on parameter estimation accuracy (≈5%), with in silico and in vivo experiment validations. This reduction of the number of needed time points by almost an order of magnitude allows the use of FLIM-FRET for certain high-throughput applications which would be infeasible if the entire number of time sampling points were used.

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Calibration Approach for Fluorescence Lifetime Determination for Applications Using Time-Gated Detection and Finite Pulse Width Excitation

Cited by 4 publications

References 23 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

Temporal Data Set Reduction Based on D-Optimality for Quantitative FLIM-FRET Imaging

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