Date Published:

10 Dec, 2023

Abstract:

Super-resolution light microscopy techniques facilitate the observation of nm-sized 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 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 analysis of the simulation results reports the conditions in which true-positive rates are maximal. We then show the capabilities of FRETsael on simulated samples of actin-vinculin and ER-ribosome interactions, as well as experimental samples of actin-myosin two-color confocal imaging. Overall, the FRETsael approach paves the way toward studying biomolecular interactions with improved spatial resolution from laser scanning confocal two-color fluorescence lifetime imaging.

Publisher's Version