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Frequently Asked Questions

Q. I accidentally deleted a molecule. Can I get it back?

Deleted molecules are saved in the recycle bin. To get back a deleted molecule, see this documentation page.

Q. I have a sample with very high FRET efficiency and no donor signal. How do I detect FRET-pairs?

For very high FRET efficiency samples you can define a FRET-pair at every detected acceptor position - without having found any donor signal at that position (you can do the opposite for very low FRET efficiency samples). Go to Settings->Peak finder settings and select 'Put a D at every A position' (in the bottom of the dialog). Select OK. Note that you will have to align the two emission channels on an alignment sample with lower FRET efficiency before finding the FRET pairs. How to use emission channel alignment on another sample is described here.

Q. I don't know what my camera background is. Which camera background settings should I use then?

Use the pixel inspection tool in iSMS to get an idea of your camera offset value. If you are still in doubt we recommend not to apply any camera background subtraction in this case. Intrinsic EMCCD camera backgrounds are typically uniform and the camera background subtraction does not have an influence on the calculated FRET efficiency. The camera background setting has an influence on the magnitude of the calculated raw background signal - your background signal and raw, background-uncorrected fluorescence intensities will appear higher than they actually are. See this documentation page for more information on camera background subtraction.

Q. How do I automatically align the emission channels?

This is done using the auto-align button located right above the raw image frame in the main window. See this documentation page for more details.

Q. Can I include auto-alignment of emission channels in the automated analysis run?

Yes. Go to 'Settings->Autorun settings' and select 'Auto-align ROIs as a first step'.

Q. How do I convert the time-series time-scale from frames to seconds?

To apply a time scale in units of second to all time-series and time-trace analysis plots in iSMS, you must set the integration time of each movie file in the file settings menu of the main window: Go to File->File settings. The integration time of each file is specified as ms/frame. If an integration time has been defined for a given file, iSMS will automatically convert all time scales to seconds. In ALEX the time-step in between each donor-excitation frame is 2*integration time.

Q. Is it possible to do analysis on single-color movies (for example, only Cy3 or only Cy5)? 

Analyzing single-color, non-FRET movies is currently not a documented option in iSMS. However, to perform this kind of analysis we suggest to do the following workaround:

  1. Select single-color excitation scheme
  2. Load the single-fluorophore movies
  3. Set the size and position of the two emission channels so that they overlap completely in the raw image frame to the left (e.g. by covering the entire image frame)
  4. Run analysis
  5. Open the FRET-pairs trace window: the top plotted traces will be the single fluorophore intensity traces
  6. Export the data