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