FRETBursts: An Open Source Toolkit for Analysis of Freely-Diusing Single-Molecule FRET

Single-molecule Förster Resonance Energy Transfer (smFRET) allows probing intermolecular interactions and conformational changes in biomacromolecules, and represents an invaluable tool for studying cellular processes at the molecular scale. smFRET experiments can detect the distance between two ƒuorescent labels (donor and acceptor) in the 3-10 nm range. In the commonly employed confocal geometry, molecules are free to di‚use in solution. When a molecule traverses the excitation volume, it emits a burst of photons, which can be detected by single-photon avalanche diode (SPAD) detectors. Œe intensities of donor and acceptor ƒuorescence can then be related to the distance between the two ƒuorophores. While recent years have seen a growing number of contributions proposing improvements or new techniques in smFRET data analysis, rarely have those publications been accompanied by so‰ware implementation. In particular, despite the widespread application of smFRET, no complete so‰ware package for smFRET burst analysis is freely available to date. In this paper, we introduce FRETBursts, an open source so‰ware for analysis of freely-di‚using smFRET data. FRETBursts allows executing all the fundamental steps of smFRET bursts analysis using state-of-the-art as well as novel techniques, while providing an open, robust and welldocumented implementation. Œerefore, FRETBursts represents an ideal platform for comparison and development of new methods in burst analysis. We employ modern so‰ware engineering principles in order to minimize bugs and facilitate long-term maintainability. Furthermore, we place a strong focus on reproducibility by relying on Jupyter notebooks for FRETBursts execution. Notebooks are executable documents capturing all the steps of the analysis (including data €les, input parameters, and results) and can be easily shared to replicate complete smFRET analyzes. Notebooks allow beginners to execute complex workƒows and advanced users to customize the analysis for their own needs. By bundling analysis description, code and ∗[email protected] results in a single document, FRETBursts allows to seamless share analysis workƒows and results, encourages reproducibility and facilitates collaboration among researchers in the single-molecule community.