Subtract module


Hello –

I am just acquainting myself with CellProfiler (great program!) and have a question about the settings for the subtract module. I would like to use an image as a negative control and subtract the average intensity of that image from the intensities of subsequent images. Ultimately, I would like to generate intensity histograms for images that have been corrected in this manner. I see that there is a subtract module to perform this task but am having trouble instructing the module to make this adjustment – I assume I’m not providing the right commands. I appreciate your help on this matter.

Thank you,



To address your problem, please post the error that you are receiving.



Hi Martha –

To process the image I would like to use as a negative control, I have been thresholding the image, measuring the intensity, and generating the intensity histogram. I would then like to process subsequent images in the same fashion, only this time using the subtract module to correct for the intensity found for the negative control.

However, in the subtract module, where I specify “subtract this image…from this image,” the only images I’m able to select are the current and thresholded images, but not a previously analyzed image (the negative control).

Is this a question of me having named the files inappropriately or having placed them in the incorrect folder (right now, all images are in the default image/output folder)?

Thank you for your help – it’s much appreciated!



Hi Erin,
Have you tried adding the LoadSingleImage module? This will allow you to load your negative control image to your current pipeline.