Single-Molecule FRET Microscopy Software

Aarhus University Seal
 iSMS About Research Groups Single molecule biophysics and chemistry group Software Documentation Export Data and Figures Export to other smFRET software

Exporting data pre-formatted for external smFRET software

You can export the time series produced using iSMS as pre-formatted input files for other more specialized smFRET software:

For HMM dwell-time analysis you can try out ebFRET, vbFRET, QuB, SMART and/or HaMMy. Note that iSMS already implements HMM analysis by variational Bayesian expectation maximization (the algorithm used in vbFRET). See Quick Guide 3 for more information on how to analyse transitional dynamics using HMM in iSMS.

For thermodynamics and kinetics analysis you can try out SMART and BOBA FRET.

Other export options of the molecule data from iSMS include generic ASCII format, the Single-Molecule Dataset (SMD) format and MATLAB structures.

In iSMS, go to the export menu and select the export format.
In the dialog, specify the molecules you wish to export. To export a specific set of molecules, select these in the FRET-pairs trace window and select 'All molecules selected in the FRET-pairs traces window'.

Export for ebFRET and SMART

ebFRET is a successor of vbFRET that analyses multiple FRET traces at once. Ref: Jan-Willem van de Meent, Jonathan E. Bronson, Chris H. Wiggins, Ruben L. Gonzalez Jr. "Empirical Bayes methods enable advanced population-level analyses of single-molecule FRET experiments", Biophys. J. (2014), 106, 6, p1327–1337

SMART is an smFRET analysis toolkit developed in the Herschlag lab at Stanford. Ref: Greenfeld, Max et al. “Single Molecule Analysis Research Tool (SMART): An Integrated Approach for Analyzing Single Molecule Data.” Ed. Daniel J. Muller. PLoS ONE 7.2 (2012): e30024. PMC. Web. 18 Mar. 2015.

Both ebFRET and SMART supports the Single-Molecule Dataset (SMD) format. Ref: Max Greenfeld, Jan-Willem van de Meent, Dmitri S Pavlichin, Hideo Mabuchi, Chris H Wiggins, Ruben L Gonzalez and Daniel Herschlag, "Single-molecule dataset (SMD): a generalized storage format for raw and processed single-molecule data", BMC Bioinformatics (2015), 16:3

First export the data from iSMS to SMD (mat).

In ebFRET, select File->Load data
In the file selection dialog, select the 'SMD time series (.mat)' format. Select the exported file and click OK.
In the dialog, select either 'Donor-Acceptor', 'donor' and 'acceptor' in the popup menus, or, 'FRET' and 'fret'.
The exported molecules are now loaded into ebFRET for further analysis.

Export for vbFRET

vbFRET is a software that models individual FRET traces using hidden Markov modeling (HMM) and finds the most probable idealized trace using evidence maximization and the variational Bayesian expectation maximization algorithm (VBEM). Reference: Bronson J.E., Fei J., Hofman J.M., Gonzalez R.L., Wiggins C.H. “Learning Rates and States from Biophysical Time Series: A Bayesian Approach to Model Selection and single-molecule FRET Data”. Biophysical Journal 97(12)3196-3205, 2009

In vbFRET, select File->Load data.
Click Add files and select the file you exported from iSMS.
Click load data.
All exported molecules are now loaded into vbFRET for further analysis.

Export for HaMMy

HaMMy was the first software specifically designed for HMM analysis of smFRET traces. Ref: S.A. McKinney, C. Joo and T. Ha, "Analysis of single molecule FRET trajectories using hidden Markov modeling", Biophysical Journal (2006)

In HaMMy, press 'Load and Analyze Data'. Locate the exported files.

Export for QuB

QuB is a software package for HMM time-series analysis originally designed for ion channel experiments. Ref: Milescu, L.S., Nicolai, C., Bannen, J., 2000-2013 QuB Software.

For analysis using QuB export the traces from iSMS as regular ASCII-files (txt).

In QuB Express, select Data->Open.
Locate the exported ascii-file.
Select as samples data.
Specify the "sampling frequency" corresponding to the inverse of your timescale in seconds.
The data is now loaded into QuB for further analysis.

Export for BOBA FRET

BOBA FRET is a software designed to quantify the cross-sample variability in smFRET data. Ref: Sebastian L. B. König, Mélodie Hadzic, Erica Fiorini, Richard Börner, Danny Kowerko, Wolf U. Blanckenhorn, Roland K. O. Sigel*,BOBA FRET: Bootstrap-based analysis of single-molecule FRET data. PLoS ONE 8(12): e84157


Export molecules from iSMS as hist2 files (single-molecule FRET histograms). When exporting from iSMS you must specify the number of bins and lower and upper bounds for each smFRET histogram (or use the default).

In BOBA, select file extension hist2 and specify the folderpath to the directory containing the exported files.
Press start RMSE analysis.
Select Yes.
The data is loaded into the program for further analysis.
Revised 17.04.2023