Images with two regions of background


I am a new user of cellprofiler and love it. I have been using this to quantity cell number by nuclear counting, among other uses. Recently, I have acquired some images using a high content imaging system and the images contain a well region with nuclear stained cells and a region outside the well (please see the attached image). This thus creates two backgrounds, one inside the well and one outside the well. My pipeline worked well with images that don’t contain this extrawell space but doesn’t work on images with a second background. I don’t know how to tell the pipeline to regard that as background. Help please!
Thank you very much!

Hi Sally,

Often, the region outside the well is either dimmer or brighter than inside the well, such that you can create a mask to mask out the exterior region. In your case here, it is about the same intensity in vs. out. So, you could possibly take a bright-filed image and do the procedure I mentioned above, or likely better and certainly easier, is to enhance the objects of interest and then the problem of the well becomes moot. That is assuming you only care about the cells here.

In the attached pipeline, I simply enhanced objects on the size scale of the cells, and thus suppressed the regions with no variation in intensity. Does this do well enough for you? If some of the cells are being suppressed too much, then make the EnhanceOrSuppressObjects scale bigger.

Hope that helps,
DL_enhance_pipeline.cppipe (3.53 KB)