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bioRxiv · 10.1101/2023.12.10.570984

FRET-sensitized acceptor emission localization (FRETsael) - nanometer localization of biomolecular interactions using fluorescence lifetime imaging

Razvag, Y.·Drori, P.·Klemfner, S.·Meshorer, E.·Lerner, E.
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Abstract

Super-resolution light microscopy techniques facilitate the observation of nanometer-size biomolecules, which are 1-2 orders of magnitude smaller than the diffraction limit of light. Using super-resolution microscopy techniques, it is possible to observe fluorescence from two biomolecules in close proximity, however not necessarily in direct interaction. Using FRET-sensitized acceptor emission localization (FRETsael), we localize biomolecular interactions exhibiting FRET with nanometer accuracy, from two-color fluorescence lifetime imaging data. The concepts of FRETsael were tested first against simulations, in which the recovered localization accuracy is 20-30 nm for true-positive detections of FRET pairs. Further analyses of the simulation results report the conditions in which true-positive rates are maximal. We then show the capabilities of FRETsael on simulated samples of Actin-Vinculin and ER-ribosomes interactions, as well as on experimental samples of actin-myosin two-color confocal imaging. Conclusively, the FRETsael approach paves the way towards studying biomolecular interactions with improved spatial resolution from laser scanning confocal two-color fluorescence lifetime imaging. SignificanceFRET is used in fluorescence microscopy to report whether dye-labeled biomolecules of choice are close within distances of 10 nm or less, hence typical interaction distances. However, in many cases, using FRET imaging for the study of biomolecular interactions is difficult due to the high density of dye-labeled biomolecules and due to the existence of unbound dye-labeled biomolecules. In addition, the resolution of localizing molecules using light microscopy is diffraction limited. This work presents FRETsael, a new approach for localizing interacting biomolecules undergoing FRET, with improved resolution of 20-30 nm for confocal microscopy using search algorithms for local extrema in contribution to FRET using two-channel fluorescence intensity and lifetime data.

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Razvag, Y., Drori, P., Klemfner, S., Meshorer, E., Lerner, E.. 2023-12-10. FRET-sensitized acceptor emission localization (FRETsael) - nanometer localization of biomolecular interactions using fluorescence lifetime imaging. https://doi.org/10.1101/2023.12.10.570984

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