It is important to avoid saturation effects. Saturation occurs when grey levels exceed the maximum available. When a spot becomes saturated, any differences in high pixel intensities cannot be resolved, and the spot appears truncated when viewed in 3D.
Why can saturation be a problem? The outline detection is not optimised for the unnatural shape of these spots, since the peaks are effectively ‘missing’ in the scanned image, and no reliable quantitation can be done with them. Saturation can cause issues in spot detection. It also prevents accurate quantitation of the spots as the true peak height cannot be known.
To find out more, download our image capture guide: http://totallab.com/cms/pdf/imagecaptureebook.pdf