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Subtraction of Camera Background in iSMS

About camera background in iSMS

The camera background depends on your camera and is preferably subtracted before loading the data into iSMS.

In iSMS, a camera background is subtracted from the raw image data when the file is loaded. The camera background can not be changed after loading the data - i.e. if you change the camera background settings you must reload the raw file again with the new settings.

Note that pixel values resulting in negative values after camera background subtraction are set to 0. This is discussed in the memory management documentation. It is therefore important that you don't use a value for the camera background larger than the actual value.

Camera background settings

Camera background types

Measured backgroud: Measured backgrounds can only be used if the raw data format is .sif (a raw file format of Andor cameras).

Offset: Many cameras have a constant offset of e.g. 100 to avoid negative values in the raw data. With a constant offset of 100, the 'negative values' in the raw data correspond to all pixel values below 100. If you select a constant offset value, specify the value in the edit box.

If in doubt, do not use camera subtraction. The camera background does not have an effect on the measured FRET efficiencies. Indeed for intensity integration iSMS uses local background subtraction for each individual molecule.