Subtracting ROIs

Hello everyone!
I’m a new user here, and relatively new (and inexperienced) when it comes to ImageJ analysis. I’m currently trying to analyze distribution of molecules between nucleus and ctyoplasm and my logic was to draw two ROIs: one for the nucleus, and one for the whole cell, then subtract the values. However, I cannot find a way to actually exclude the nuclear ROI from the whole cell ROI in ImageJ (to only measure the cytoplasm), so if you know of an ImageJ function to do that, don’t hesitate to share!
Thank you all in advance!

Hello caffeinated -

Let’s assume that your nuclear ROI is contained entirely within
your cell ROI. Add both ROIs to the ROI Manager. Select both
ROIs in the ROI Manager (or deselect both), and, from the
buttons on the right-hand side of the ROI Manager, run
More >> XOR.

This will create your desired ROI on your image. You can add
it to the ROI Manager if you want.

Thank, mm


  1. Select the cytoplasm and the nuclear ROIs,
  2. in ROIs Manager, click on “More” and select “XOR”
  3. you now have a ROI that only contains the cytoplasm to the exclusion of the nucleus. Add it to the manager and measure.



Hi @mountain_man and @MatthieuV, thank you both for the help!