Tracking of smFRET data based TIRF microscopy of diffusing receptors in live cells

A Matlab script to track smFRET data obtained by the Matlab software FRET_efficiency_analysis published by Winkelmann et al. (https://doi.org/10.1038/s41467-024-49876-9). FRET efficiency analysis is a batch script to analyze multiple datasets (individual cells). It generates a table of pooled FRET efficiency and stoichiometry data for single-molecule FRET events including there localizations and time-points. This data is used here to generate trajectories based on nearest-neighbor tracking including gap closing. User needs to define the following parameters

System Requirements (tested environment)

• Operating System: Microsoft Windows 11 Home Version 23H2 (Build 22631.3527)
• Tested MATLAB Version: 9.13.0.2698988 (R2022b) Update 10

Containing files

• trackPooledEMapsWithGapClosure.m: Main script containing nearest-neighbor tracking and gap closing,
• plotEandSvst.m: Script to plot FRET efficiencies and stoichiometries versus time.

Installation guide

• Install Matlab and required toolboxes (installation time < 1 h),
• extract ZIP-file 'SPT_Simulator.zip'.

Instructions to run

1. Start Matlab 2022b,
2. In Matlab browser, navigate to software directory (...\smFRET_tracking),
3. Open 'trackPooledEMapsWithGapClosure.m' in Matlab, set parameters to your needs and run. After starting the script, user need to load txt file containing pooled E-S table and define folder to save results.
4. Open 'plotEandSvst.m' in Matlab, set parameters to your needs and run.

Expected run time with default parameters less than 1 minute, using a computer with AMD Ryzen 7 4700U or similar.

Input parameters

• D: rough estimate of the 2D diffusion constant of the molecules to track.
• dt: Temporal resolution of input data.
• nFrGapClosure: Maximum number of frames to consider for gap closing.
• pxlSz: Pixel size of raw data.
• nPxlX: Horizontal resolution of input images in pixels.
• nPxlY: Vertical resolution of input images in pixels.
• trLenForPlot: Minimal track length for final plotting of trajectories

Main outputs:

• particleTracks: Structure array with all tracks without considering gap closing. Each field (row) is a full track with 12 entries (columns): cell #, track id, time [s], x [µm], y [µm], FRET efficiency E_raw, FRET efficiency EPR, corrected FRET efficiency E, stoichiometry S_raw, stoichiometry SPR, gamma corrected stoichiometry S_gamma, gamma and beta corrected stoichiometry S_gammabeta
• particleTracksGapClosure: Same structure array as particleTracks, but considering gap closure to assemble final tracks
• Figure showing all trajectories above user defined track length threshold.
• mat file containg all variables.

Author

Rainer Kurre, PhD

Email: rainer.kurre@uos.de

Center for Cellular Nanoanalytics and Division of Biophysics

Osnabrueck University, Barabastr. 11, D-49076 Osnabrück %Center for Cellular Nanoanalytics and Division of Biophysics %Osnabrueck University, Barabastr. 11, D-49076 Osnabrück