How to Subtract two images created from Oval ROIs?

[kloram333]

I am trying to subtract two images that have each been created from a user-selected oval shaped ROI. I can preform the image subtraction using the original images, and then selecting an ROI (I can only get rectangular ROIs to work) from the resulting subtracted image and converting that ROI to an image. I'll attach an example program and some sample images for clarity. Any suggestions would be greatly appreciated!!

[NECO2012]

Don't quite understand your question.  Your VI uses ROI to Image (I assume you downloaded from https://decibel.ni.com/content/docs/DOC-9564).  That VI uses Convert ROI to Rectangle which makes sense why you can only get rectangular ROIs to work.  Also no example images are attached.

[NECO2012]

Please see attached image.  ROI In Descriptor not needed.  I just did not want to take the time to size them both the same each time. Also breakpoint was so I could use trash all for images but still make sure the image is correct.  You will get an oval subtracted image on a black border.  This is due to there being no transparency in grayscale.