FRET-sensitized acceptor emission localization (FRETsael) – nanometer accuracy localization of biomolecular interactions using FLIM-FRET laser scanning confocal microscopy

Super-resolution light microscopy techniques shifted the diffraction-limited microscopy into nanoscopy, in which it is finally possible to observe the nanometer-scale biomolecules in the cell. Attaining the super-resolved information requires either using fluorescent dyes flickering between bright and dark states, immediate depletion of a well-defined region of dye excited states or using the knowledge of the illumination profile to track single biomolecules. We developed a technique dubbed FRET-sensitized acceptor emission localization (FRETsael), in which we gain nanometer localization accuracy of biomolecular interactions in FRET-FLIM. This is achieved by few nanometer laser scanning steps remembering the confocal illumination profile is spatially non-uniform illumination, and by finding the local extrema of parameters that report on the contribution of excitation to FRET. Using simulations, we show that no the localization accuracy is 20-30 nm for all true-positive detections no matter what are the underlying experimental conditions. We also report the dynamic range in which the false discovery rate is minimal, and the true positive rate is maximal. Furthermore, we show the performance of the algorithm on a more realistic simulations of Actin-Vinculin and ER-Ribosomes pairs of interactions. Finally, we explore the performance of the FRETsael approach on cells with the nuclear pore protein Nup96 tagged with a donor GFP and with Alexa Fluor 647-labeled antibodies against GFP, as acceptors. The FRETsael imaging approach paves the way towards studying biomolecular interactions with improved spatial resolution from alternating laser excitation scanned frames in confocal microscopy without the use of blinking dyes or special optics.