Single-Molecule FRET Microscopy Software

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Compensate drift in TIRF microscopy movies

About drift compensation

Sample drift is often a problem in single-molecule FRET TIRF-microscopy when molecules drift over distances >1 pixel through the recorded movie.

iSMS can be used to compensate horizontal sample drift when all molecules are subject to the same drift. The program analyses drift using a discrete Fourier transform (DFT) based algorithm for translational image registration between a reference image (an average of a certain frame interval) and all frames throughout the movie. The drift is then compensated by translating all frames in the movie a distance (x,y) defined by the detected shift vector.

Note that drift compensation in iSMS will create a new drift-compensated movie which takes up large amounts of RAM. See this page for managing memory issues in iSMS.

Analyse drift

Open the drift window in 'Tools->Drift analysis'.
In the drift window, press 'Analyse drift'. This will run the drift analysis on the selected files.
The result of the drift analysis is plotted in the two plots. The left plot shows the direction of drifting. Red indicates the beginning of the movie, green the middle of the movie and blue the end of the movie. The right plot shows the magnitude of drifting as a function of time.

Compensate drift

To compensate for the analysed drift select the 'Apply drift compensation' checkbox. This will start the drift compensation. When finished, you can see the effect of the drift compensation on individual molecule traces in the lower part of the window. In the trace plots, black curves show the traces before drift compensation and colored traces are after drift compensation.

Simulate synthetic drift

Synthetic drift was originally implemented in iSMS to test the drift compensation algorithm. Now, synthetic drift may be useful for testing and educational purposes.
Go to 'Tools->Simulate synthetic drift'.
Specify the amount of drift in the dialog and press OK.
A synthetic drift is now applied to the selected movies. After a new drift analysis of the movies, the applied drift is clearly seen in the drift plots.
Now see the effect of drift compensation of the synthetically applied drift by selecting the 'Apply drift compensation' check box. After compensation, the black traces show the drifted molecule and colored traces show the drift compensated molecule.
Revised 17.04.2023