Colocalization: Segmentation and Exporting Child/Parent data

Sample image and/or macro code


I am new to Cell Profiler, and I am looking to use it to analyze colocalization of trafficking proteins with an intracellular pathogen. I have adjusted the colocalization example from the online tutorials to try to create a pipeline for this project.

The pipeline I am currently working with (images included):7.17.20 Test.cpproj (131.8 KB)


These images have Macrophages:labeled in DAPI, Pathogen: labeled in FITC, and Trafficking proteins: Alx647.

Analysis goals

The goal is to be able to determine how many colocalization events are occurring in each image. So ideally, the ultimate output would be: # of colocalized pathogen/ # total pathogen.


-I have tried to troubleshoot the segmentation of the pathogen by shape, intensity and size, but sometimes it segments 1 pathogen into multiple objects. Is there another way adjust the segmentation?

-Is there a way to export the parent/children values in order to get a spreadsheet in which I could calculate: # of colocalized pathogen/ # total pathogen

-What is the benefit of including the expand/shrink modules in a colocalization pipeline?

  • I am not sure whether the correct illumination calculate has the correct settings for these image. If there is further information as to help the user choose which is the most optimal settings, please let me know.


Hi @micro,

Possible answers to your challenge,
For segmentation, you can try adjusting the declumping value.
I think you can export the children count using the “ExportSpreadSheet” based on the parent object. In case you want to compute the ratio you could try “CalculateMath” module.
Unless there is a sample image it would be difficult to help with illumination correction. In case you need further help, you might have to share the screenshot of the error or sample image.