FRETBursts

FRETBursts analyzes freely-diffusing single-molecule Förster Resonance Energy Transfer (smFRET) data to detect and process fluorescence photon bursts and compute FRET efficiencies for studying intermolecular interactions and conformational dynamics.

Key Features:

• Freely-diffusing smFRET (confocal): Supports analysis of confocal-geometry experiments where molecules diffuse through an excitation volume.
• Photon burst detection: Identifies photon bursts produced as molecules traverse the excitation volume from single-photon avalanche diode (SPAD) detector time traces.
• Donor and acceptor fluorescence analysis: Analyzes donor and acceptor fluorophore intensities to compute FRET efficiencies and infer inter-dye distances.
• Distance sensitivity: Operates on FRET-measured distances in the typical 3–10 nanometer range between donor and acceptor labels.
• Complete burst-analysis suite: Implements the fundamental steps required for smFRET burst analysis across detection and quantification stages.
• Methodological breadth: Incorporates both state-of-the-art and novel methodologies for robust burst analysis.
• Reproducible, notebook-based workflows: Uses Jupyter notebooks to encapsulate data files, input parameters, and results for reproducible computational analyses.
• Robustness and maintainability: Emphasizes software engineering practices to reduce bugs and support long-term maintainability of analysis code.

Scientific Applications:

• Intermolecular interactions: Quantifies FRET efficiencies to study interactions between biomolecules labeled with donor and acceptor fluorophores.
• Conformational dynamics: Infers conformational changes and distance distributions within biomacromolecules from FRET-derived distances (3–10 nm).
• Method development and benchmarking: Provides a platform for comparison, validation, and development of new smFRET burst analysis methods.

Methodology:

Detects photon bursts from SPAD detector time traces in confocal freely-diffusing smFRET experiments and analyzes donor and acceptor fluorescence intensities to compute FRET efficiencies and infer inter-dye distances, with workflows encapsulated in Jupyter notebooks.