Improving bi-exponential fitting of autofluorescence lifetime decay data through whole-cell binning

SignificanceFluorescence lifetime imaging (FLIM) analysis commonly involves multi-exponential fitting of fluorescence decays, which requires relatively high signal-to-noise ratios (SNRs). However, autofluorescence presents a weaker signal compared with exogenous fluorophores, which can make fitting challenging. Becker & Hickl’s SPCImage (SPCI) is commonly used for bi-exponential fitting of NAD(P)H autofluorescence decays, but its accuracy has not been robustly evaluated.AimWe evaluate the accuracy of bi-exponential modeling of NAD(P)H FLIM data from live cells and provide an alternative fitting algorithm that allows for photon binning of whole cells for low SNR applications.ApproachSimulated fluorescence lifetime decay data and NADH solutions were used to test the accuracy of fitting in both SPCI and a custom-written MATLAB algorithm. FLIM images collected from live cells using varying integration times were also evaluated.ResultsResults show consistently higher error for SPCI in fluorescence decay parameters compared with the custom-written MATLAB algorithm. Whole-cell binning greatly improved the accuracy of all lifetime decay parameters, allowing for shorter integration times during acquisition.ConclusionsOur custom-written MATLAB algorithm can provide a flexible alternative fitting tool that allows for photon binning of whole cells and substantially more accurate parameter estimation.