|  Very precise background correction can be achieved by using Selected ROI. Select a ROI representing the area that was supposed to be black but absorbed some fluorescence and perform background correction. To confirm that background correction was done properly (all irrelevant pixels were removed), use Histogram. Select a channel, for example Red, and check whether any pixels of this channel remain in the selected ROI after background correction. If some of them are still in the selected ROI, use Threshold Value to remove them completely. Perform this procedure for Green and Blue channels as well. After you see that no any irrelevant pixels remain, you have corrected background properly and may start calculating colocalization coefficients.
When correcting background using selected ROI, there is no need to reset image every time when you want to repeat correction. Simply select a new ROI and perform background correction again. | |
If you wish to analyze colocalization in a specific area of the image, choose the ROI tool that will enable the most precise selection of the area you will be examining. If you find it is difficult to select an accurate ROI using rectangle and oval tools, try polygon or lasso instead.
To view colocalized pixels, click 
Although Pearson's correlation coefficient (Rr) is a CoLocalizer Pro default coefficient, we recommend paying close attention to Manders' overlap coefficient (R), as it indicates a true degree of colocalization. Manders' overlap coefficient (R) may be actually more suitable if your sections are not freshly stained and/or your images were obtained after re-examining samples.