Graphical representation and multicomponent analysis of single‐frequency fluorescence lifetime imaging microscopy data

Summary Graphical representation of fluorescence lifetime imaging microscopy data demonstrates that a mixture of two components with single exponential decays can be resolved by single frequency measurements. We derive a method based on linear fitting that allows the calculation of the fluorescence lifetimes of the two components. We show that introduction of proper error‐weighting results in a non‐linear method that is mathematically identical to a global analysis algorithm that was recently derived. The graphical approach was applied to cellular data obtained from a lifetime‐based phosphorylation assay for the epidermal growth factor receptor and yielded results similar to those obtained by a global analysis algorithm.