Comparing different modalities of time-resolved two-photon fluorescence measurements for FRET analysis

Different modalities of time-resolved fluorescence lifetime techniques are typically used for Förster resonance energy transfer (FRET) studies in vivo and in solution and have been used to measure macromolecular processes and interactions. Here, we examine the sensitivity of the estimated FRET efficiency and donor-acceptor distance to different modalities of time-resolved two-photon (2P) fluorescence measurements, experimental design, and data analysis. As a model, we used the biosensor (mTurquoise-linker-mCitrine), where mTurquoise-mCitrine serves as the FRET pair tethered with a flexible linker.