A FLIM Microscopy Based on Acceptor-Detected Förster Resonance Energy Transfer

Delgadillo, Roberto F.; Carnes, Katie A.; Zaleta-Rivera, Kathia; Olmos, Omar; Parkhurst, Lawrence J.

[Image: see text] Time-resolved donor-detected Förster resonance energy transfer (trDDFRET) allows the observation of molecular interactions of dye-labeled biomolecules in the ∼10–100 Å region. However, we can observe longer-range interactions when using time-resolved acceptor-detected FRET (trADFRET), since the signal/noise ratio can be improved when observing the acceptor emission. Therefore, we propose a new methodology based on trADFRET to construct a new fluorescence lifetime microscopy (FLIM-trADFRET) technique to observe biological machinery in the range of 100–300 Å in vivo, the last frontier in biomolecular medicine. The integrated trADFRET signal is extracted in such a way that noise is canceled, and more photons are collected, even though trADFRET and trDDFRET have the same rate of transfer. To assess our new methodology, proof of concept was demonstrated with a set of well-defined DNA scaffolds.