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What does the subtract background command do?
Removes smooth continuous backgrounds from gels and other images. Based on the a “rolling ball” algorithm described in Stanley Sternberg's article, “Biomedical Image Processing”, IEEE Computer, January 1983. Imagine a 3D surface with the pixel values of the image being the height, then a ball rolling over the back side of the surface creates the background. The current algorithm (since version 1.39f) uses an approximation of a paraboloid of rotation instead of a ball.
The Rolling Ball Radius is the radius of curvature of the paraboloid. As a rule of thumb, for 8-bit or RGB images it should be at least as large as the radius of the largest object in the image that is not part of the background. Larger values will also work unless the background of the image is too uneven. For images with pixel values having a very different range, note that the radius should be inversely proportional to the pixel value range. E.g., typical values of the radius are around 0.2 to 5 for 16-bit images (pixel values 0…65535).
With the Create Background option, the output is not the image with the background subtracted but rather the background itself. This option is useful for examining the background created (in conjunction with the Preview option). “Create Background” can be also used for custom background subtraction algorithms where the image is duplicated and filtered (e.g. removing “holes” in the background) before creating the background and finally subtracting it with Process > Image Calculator.
For calculating the background (“rolling the ball”), images are normally smoothened to reduce noise (average over 3×3 pixels). With Disable Smoothing, the unmodified image data are used for creating the background. Check this option to make sure that the image data after subtraction will never be below the background.