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 is coming from adjacent scanned planes and 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 coefficients 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 Selected ROI. In this case, a region of interest (ROI) is selected within the image background. Based on the riginally-developed algorithms, pixels of selected values are then subtracted from the pixel profile of the whole image. Resulted image thus becomes suitable for coefficients calculations.
