Reliability of coefficients calculations depends on the quality of analyzed images. Unfortunately, even perfectly acquired confocal fluorescence microscopy images can not be used for quantification purposes without adjustments, because they contain a certain amount of out-of-focus light. This light comes from adjacent scanned planes and is manifested as a background noise (pixels of "uncertain" values) in the images. Background noise is especially ominous when analyzed images are of low contrast and/or have weak fluorescence. If not removed, pixels of these values may interfere with execution of the implemented algorithms and cause erroneous readings. It was reported that the use of unadjusted images may result in up to 30% overestimation of colocalization.

In most cases, correcting background in Auto mode and selecting image pattern Average Contrast and Fluorescence will produce acceptable and reproducable results.

More precise and individually image-tailored correction can be achieved in Manual mode using the Selected ROI. In this case, a region of interest (ROI) is selected within the image background. Pixels of the selected values are then subtracted from the pixel profile of the whole image. Resulted image thus becomes suitable for coefficients calculations.