Nucleolus Pipeline Problems



Hi Guys, I have some questions for you and I will appreciate any help you can provide. During few weeks I have built several pipelines that have failed and I have started to think either it might not be possible to do it with CP or I’m making some basic mistake. I want to build a pipeline that messure a few parameters at the nucleolus, nucleoplasm and nucleous (intensity of an interest protein and fibrillarin, colocalization with nucleolar marker, granularity, so on). For these I have used several approaches without any success. To detect nucleolus I have a nucleolar marker, fibrillarin, which I used to detect as primary object -Im also intersted in meassure area, intensity, so on- (Nucleolo_object), then I detect Nucleus using DAPI and I use it as primary object (Nucleo). Then used the module MaskObject to mask the nucleolus and finally I used the module measureObjectIntensity to see whether is working. the problem is that regardless the combination I used in the masking, the intensities (integrated intensity) of DAPI and Fibrillarin, don’t make any sense (the nucleolus should have almost all the Fibrillarin and non DAPI, and the nucleus should have all DAPI and all fibrillarin too), I’m confuse, since I don’t know what is the softwere reading or if maskingObject is the right module. In addition, I tried to use the nucleous as secondary object, but it did not do the segmentation properly. Can you suggest a workflow? Any help will be highly appreciate it. Regards.
Nucleolus Pipeline.cppipe (11.4 KB)



So the question was:

  • You want to detect nucleoli using nucleolar marker, fibrillarin, as primary object (Nucleolo_object),
  • You want to dected nucleus using DAPI, as primary object (Nucleo).
  • Then measure everything inside the detected objects Nucleo and Nucleolo_object.

In your current pipeline, the problem was the module MaskObject. You do not need to mask anything to measure the object intensity. You can just add module MeasureObjectIntensity right after IdentifyPrimaryObjects, then simply select the detected objects and the channels.

“In addition,… use the nucleous as secondary object, but it did not do the segmentation properly.”

  • You should use RelateObjects instead of IdentifySecondaryObjects. In RelateObjects >> choose Nucleus as parent, Nucleolus as child.

Hope that helps.