I am working on a pipeline which identifies child objects inside of the nucleus. The pipeline currently set to where C.P. identifies the nucleus and I manually select which nuclei I would like to keep. The objects I decide to keep are then cropped or masked (I’ve tried both methods) from the original image. The threshold of each nuclei is analyzed on an otsu per object bases and immunofluorescent objects (foci) are identified. But the problem is that the intensity of the nuclei is altered when it is cropped and placed onto a new background. What I observe is that the background noise of the nuclei becomes pronounced and the program has a difficult time distinguishing background from the immunofluorescent objects. I attached an image to help describe the problem. Is there any way around this? Is there any way we can revert the illumination of the cropped images back to the intensity of the original image? I would appreciate any help.