title: Reduced temporal sampling effect on accuracy of time-domain fluorescence lifetime Förster resonance energy transfer.
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).
authors.affiliation: Rensselaer Polytechnic Institute, Department of Biomedical Engineering, 110 8th Street, Troy, New York 12180, United States.
authors.forename: Travis
authors.initials: T
authors.lastname: Omer
authors.affiliation: Rensselaer Polytechnic Institute, Department of Biomedical Engineering, 110 8th Street, Troy, New York 12180, United States.
authors.forename: Lingling
authors.initials: L
authors.lastname: Zhao
authors.affiliation: Rensselaer Polytechnic Institute, Department of Biomedical Engineering, 110 8th Street, Troy, New York 12180, United States.
authors.forename: Xavier
authors.initials: X
authors.lastname: Intes
authors.affiliation: Rensselaer Polytechnic Institute, Departments of Biomedical Engineering and Chemical & Biological Engineering, 110 8th Street, Troy, New York 12180, United States.
authors.forename: Juergen
authors.initials: J
authors.lastname: Hahn
mesh_terms: D000465:Algorithms; D000818:Animals; D016477:Artifacts; D003627:Data Interpretation, Statistical; D004285:Dogs; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D007089:Image Enhancement / Q000379:methods; D007090:Image Interpretation, Computer-Assisted / Q000379:methods*; D061985:Madin Darby Canine Kidney Cells; D008856:Microscopy, Fluorescence / Q000379:methods*; D057054:Molecular Imaging / Q000379:methods*; D015203:Reproducibility of Results; D018401:Sample Size; D012680:Sensitivity and Specificity