FRETBursts: An Open Source Toolkit for Analysis of Freely-Diffusing 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 fluorescent labels (donor and acceptor) in the 3-10 nm range. In the commonly employed confocal geometry, molecules are free to diffuse 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. The intensities of donor and acceptor fluorescence can then be related to the distance between the two fluorophores. 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 software implementation. In particular, despite the widespread application of smFRET, no complete software package for smFRET burst analysis is freely available to date. In this paper, we introduce FRETBursts, an open source software for analysis of freely-diffusing 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 well-documented implementation. Therefore, FRETBursts represents an ideal platform for comparison and development of new methods in burst analysis. We employ modern software 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 files, input parameters, and results) and can be easily shared to replicate complete smFRET analyzes. Notebooks allow beginners to execute complex workflows and advanced users to customize the analysis for their own needs. By bundling analysis description, code and results in a single document, FRETBursts allows to seamless share analysis workflows and results, encourages reproducibility and facilitates collaboration among researchers in the single-molecule community.

[1]  Elisha Haas,et al.  Resolution of Two Sub-Populations of Conformers and Their Individual Dynamics by Time Resolved Ensemble Level FRET Measurements , 2015, PloS one.

[2]  Richard J. Boys,et al.  Discussion of "Bayesian analysis of single-molecule experimental data" by Kou, Xie and Liu , 2005 .

[3]  David Willshaw,et al.  Towards standard practices for sharing computer code and programs in neuroscience , 2016 .

[4]  Antonino Ingargiola,et al.  Photon-HDF5: An Open File Format for Timestamp-Based Single-Molecule Fluorescence Experiments. , 2016, Biophysical journal.

[5]  M. Stephens.  EDF Statistics for Goodness of Fit and Some Comparisons , 1974 .

[6]  Helen Shen,et al.  Interactive notebooks: Sharing the code , 2014, Nature.

[7]  Francesco Panzeri,et al.  8-spot smFRET analysis using two 8-pixel SPAD arrays , 2013, Photonics West - Biomedical Optics.

[8]  Paul R. Selvin,et al.  The renaissance of fluorescence resonance energy transfer , 2000, Nature Structural Biology.

[9]  Suren Felekyan,et al.  Separating structural heterogeneities from stochastic variations in fluorescence resonance energy transfer distributions via photon distribution analysis. , 2006, The journal of physical chemistry. B.

[10]  Suren Felekyan,et al.  Probability distribution analysis of single-molecule fluorescence anisotropy and resonance energy transfer. , 2007, The journal of physical chemistry. B.

[11]  Daniel Barsky,et al.  Correlation spectroscopy of minor fluorescent species: signal purification and distribution analysis. , 2007, Biophysical journal.

[12]  Jun S. Liu,et al.  Bayesian analysis of single‐molecule experimental data , 2005 .

[13]  Darrel C. Ince,et al.  The case for open computer programs , 2012, Nature.

[14]  Z. Merali.  Computational science: ...Error , 2010, Nature.

[15]  Rahul Roy,et al.  A practical guide to single-molecule FRET , 2008, Nature Methods.

[16]  Yusdi Santoso,et al.  Characterizing single-molecule FRET dynamics with probability distribution analysis. , 2010, Chemphyschem : a European journal of chemical physics and physical chemistry.

[17]  Jane Clarke,et al.  Quantifying heterogeneity and conformational dynamics from single molecule FRET of diffusing molecules: recurrence analysis of single particles (RASP). , 2011, Physical chemistry chemical physics : PCCP.

[18]  Shimon Weiss,et al.  Probing structural heterogeneities and fluctuations of nucleic acids and denatured proteins. , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[19]  David L. Donoho,et al.  WaveLab and Reproducible Research , 1995 .

[20]  Marcia Levitus,et al.  Measuring conformational dynamics: a new FCS-FRET approach. , 2007, The journal of physical chemistry. B.

[21]  Antonino Ingargiola,et al.  Photon-HDF5: open data format and computational tools for timestamp-based single-molecule experiments , 2016, SPIE BiOS.

[22]  Irina V Gopich.  Concentration effects in "single-molecule" spectroscopy. , 2008, The journal of physical chemistry. B.

[23]  A Volkmer,et al.  Data registration and selective single-molecule analysis using multi-parameter fluorescence detection. , 2001, Journal of biotechnology.

[24]  Nam Ki Lee,et al.  Fluorescence-aided molecule sorting: Analysis of structure and interactions by alternating-laser excitation of single molecules , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[25]  G. Danezis,et al.  Bayesian inference of accurate population sizes and FRET efficiencies from single diffusing biomolecules. , 2014, Analytical chemistry.

[26]  Christoph Bräuchle,et al.  Pulsed interleaved excitation. , 2005, Biophysical journal.

[27]  Christian Eggeling,et al.  Quantitative identification of different single molecules by selective time-resolved confocal fluorescence spectroscopy. , 1998 .

[28]  Miran Liber,et al.  Disentangling subpopulations in single-molecule FRET and ALEX experiments with photon distribution analysis. , 2012, Biophysical journal.

[29]  E. Haas,et al.  Kinetics of fast changing intramolecular distance distributions obtained by combined analysis of FRET efficiency kinetics and time-resolved FRET equilibrium measurements. , 2014, Biophysical journal.

[30]  A. Szabó,et al.  Single-molecule FRET with diffusion and conformational dynamics. , 2007, The journal of physical chemistry. B.

[31]  G. Schwarz.  Estimating the Dimension of a Model , 1978 .

[32]  Chris H Wiggins,et al.  Empirical Bayes methods enable advanced population-level analyses of single-molecule FRET experiments. , 2014, Biophysical journal.

[33]  Chris H Wiggins,et al.  Learning rates and states from biophysical time series: a Bayesian approach to model selection and single-molecule FRET data. , 2009, Biophysical journal.

[34]  Ian M. Mitchell,et al.  Best Practices for Scientific Computing , 2012, PLoS biology.

[35]  H. Akaike.  A new look at the statistical model identification , 1974 .

[36]  Dmitri S. Pavlichin,et al.  Single Molecule Analysis Research Tool (SMART): An Integrated Approach for Analyzing Single Molecule Data , 2012, PloS one.

[37]  David Klenerman,et al.  pyFRET: A Python Library for Single Molecule Fluorescence Data Analysis , 2014, ArXiv.

[38]  A. Szabó,et al.  FRET efficiency distributions of multistate single molecules. , 2010, The journal of physical chemistry. B.

[39]  Timothy D Craggs,et al.  Alternating-laser excitation: single-molecule FRET and beyond. , 2014, Chemical Society reviews.

[40]  Per A. Brodtkorb,et al.  Lmfit/Lmfit-Py 0.9.6Rc1 , 2017 .

[41]  S. Weiss.  Fluorescence spectroscopy of single biomolecules. , 1999, Science.

[42]  R. Rigler,et al.  Conformational transitions monitored for single molecules in solution. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[43]  Danny Kowerko,et al.  BOBA FRET: Bootstrap-Based Analysis of Single-Molecule FRET Data , 2013, PloS one.

[44]  C. Seidel,et al.  Monitoring conformational dynamics of a single molecule by selective fluorescence spectroscopy. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[45]  Gilad Haran,et al.  How, when and why proteins collapse: the relation to folding. , 2012, Current opinion in structural biology.

[46]  S. McKinney,et al.  Analysis of single-molecule FRET trajectories using hidden Markov modeling. , 2006, Biophysical journal.

[47]  Nam Ki Lee,et al.  Alternating-laser excitation of single molecules. , 2005, Accounts of chemical research.

[48]  S. Gull,et al.  Classic maximum entropy recovery of the average joint distribution of apparent FRET efficiency and fluorescence photons for single-molecule burst measurements. , 2012, The journal of physical chemistry. B.

[49]  Jeremy Freeman.  Open source tools for large-scale neuroscience , 2015, Current Opinion in Neurobiology.

[50]  Christoph Deil,et al.  lmfit-py: release 0.9.3 , 2016 .

[51]  Yusdi Santoso,et al.  Identifying molecular dynamics in single-molecule FRET experiments with burst variance analysis. , 2011, Biophysical journal.

[52]  Shimon Weiss,et al.  Ratiometric measurement and identification of single diffusing molecules , 1999 .

[53]  A. Cheng,et al.  Single-molecule detection and spectroscopy in point-like geometries ( a ) Point-like excitation and detection , 2012 .

[54]  Mauno Vihinen,et al.  No more hidden solutions in bioinformatics , 2015, Nature.

[55]  Shimon Weiss,et al.  The transcription bubble of the RNA polymerase-promoter open complex exhibits conformational heterogeneity and millisecond-scale dynamics: implications for transcription start-site selection. , 2013, Journal of molecular biology.

[56]  John D. Blischak,et al.  A Quick Introduction to Version Control with Git and GitHub , 2016, PLoS Comput. Biol..

[57]  Everett A. Lipman,et al.  Förster transfer outside the weak-excitation limit , 2009 .

[58]  Kai Zhang,et al.  Photon-by-photon determination of emission bursts from diffusing single chromophores. , 2005, The journal of physical chemistry. B.

[59]  Shimon Weiss,et al.  Shot-noise limited single-molecule FRET histograms: comparison between theory and experiments. , 2006, The journal of physical chemistry. B.

[60]  B. Schuler,et al.  Unfolded protein and peptide dynamics investigated with single-molecule FRET and correlation spectroscopy from picoseconds to seconds. , 2008, The journal of physical chemistry. B.

[61]  Andreas Prlic,et al.  Ten Simple Rules for the Open Development of Scientific Software , 2012, PLoS Comput. Biol..

[62]  David A W Soergel,et al.  Rampant software errors may undermine scientific results , 2014, F1000Research.

[63]  A. Szabó,et al.  Theory of Single‐Molecule FRET Efficiency Histograms , 2011 .

[64]  M Dahan,et al.  Single-pair fluorescence resonance energy transfer on freely diffusing molecules: observation of Förster distance dependence and subpopulations. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[65]  Suren Felekyan,et al.  On the origin of broadening of single-molecule FRET efficiency distributions beyond shot noise limits. , 2010, The journal of physical chemistry. B.

[66]  David R. Anderson,et al.  Model selection and multimodel inference : a practical information-theoretic approach , 2003 .

[67]  Miran Liber,et al.  Detailed study of DNA hairpin dynamics using single-molecule fluorescence assisted by DNA origami. , 2013, The journal of physical chemistry. B.

[68]  Nam Ki Lee,et al.  Accurate FRET measurements within single diffusing biomolecules using alternating-laser excitation. , 2005, Biophysical journal.

[69]  A. Szabó,et al.  Theory of photon statistics in single-molecule Förster resonance energy transfer. , 2005, The Journal of chemical physics.

[70]  C. Seidel,et al.  Accurate single-molecule FRET studies using multiparameter fluorescence detection. , 2010, Methods in enzymology.

[71]  W. Eaton,et al.  Protein folding studied by single-molecule FRET. , 2008, Current opinion in structural biology.

[72]  A. Szabó,et al.  Decoding the pattern of photon colors in single-molecule FRET. , 2009, The journal of physical chemistry. B.

[73]  Yusdi Santoso,et al.  Probing biomolecular structures and dynamics of single molecules using in-gel alternating-laser excitation. , 2009, Analytical chemistry.