FRETsael: Nanometer localization of biomolecular interactions using fluorescence lifetime imaging

Area of Science:

• Biophysics
• Molecular Biology
• Microscopy

Background:

• Super-resolution microscopy allows observation of nanoscale biomolecules beyond the diffraction limit.
• Distinguishing close proximity from direct interaction is challenging with conventional methods.
• Fluorescence Resonance Energy Transfer (FRET) indicates proximity but requires precise localization.

Purpose of the Study:

• To develop and validate FRETsael for nanometer-accurate localization of biomolecular interactions exhibiting FRET.
• To assess the performance of FRETsael using simulations and experimental data.
• To improve the spatial resolution for studying biomolecular interactions.

Main Methods:

• Utilized FRETsael algorithm on two-color fluorescence lifetime imaging data.
• Validated the approach through extensive simulations with known FRET pairs.
• Applied FRETsael to simulated actin-vinculin and ER-ribosome interactions, and experimental actin-myosin imaging.

Main Results:

• FRETsael achieved 20-30 nm localization accuracy for true-positive FRET pair detections in simulations.
• Identified conditions for maximizing true-positive detection rates.
• Demonstrated capability in analyzing complex biological interactions like actin-vinculin and ER-ribosomes.

Conclusions:

• FRETsael accurately localizes biomolecular interactions using FRET from fluorescence lifetime imaging.
• The method enhances spatial resolution for studying molecular proximity and interactions.
• Paves the way for advanced investigations of biomolecular interactions in biological systems.