Overlay outline issues in cellprofiler! Please help! URGENT!

Sample image and/or macro codeCD45 Prim Sec AF488-Image Export-03_c1 2.tif (770.2 KB)

I’m super new to cellprofiler, still trying to figure out how to run the software and create appropriate pipeline.
To give a brief idea, I trying to create overlay outlines to my RAW cells to clearly distinguish nucleus and cytoplasm. The problem I’m facing is that, the software doesn’t recognize nucleus as it considers everything in the diameter range (10-40pixles) as cells, I have tried to reduce the range of diameter but the outline seems to broken into smaller areas.

I’m attaching files that can help.
First image- CD45 prim sec AF488 image- the original image, its a merge image for DAPI- nucleus and CD45 stained with AF488- RAW cells
Second images- This is an example pipeline and images from Cellprofiler. I’m expecting my output images to be similar to this example.
Thrid images- This is how my output images for OverLay Outline looks like now!

I’m not sure how to fix this issue, any kind of help would be very much appreciated!
Thank you in advance!!!

Hi @Isha14,

So to clarify: You want an overlay with the nuclei outlines in one colour and the cells themselves in another?

The first thing to check is whether you’re identifying nuclei and cells as separate objects? Generally we’d use the IdentifyPrimaryObjects module to find nuclei, then “grow” a cell around them using IdentifySecondaryObjects. If you’ve done that you be able to add additional outlines for these object sets in the OverlayOutlines module.

If that’s not the case, it’d be helpful if you could upload your current pipeline so that we can review the settings.


Hey @DStirling
Thank you for getting back, yes you’re right I want to overlay nuclei in one color and cells in another color.
I used the same image (merged imaged ) as mentioned above to identify nuclei as primary objects, then tried to identify secondary objects. I’m not sure what you mean by growing a cell around the nuclei. please find the pipline I used.
pipeline.cppipe (11.7 KB)

Okay, your OverlayOutlines module is set up correctly. The issue here is that your nuclear segmentation and cell segmentation aren’t all that different, so most cell outlines are on top of the nuclei outlines. By “growing” I mean that you’re using the IdentifySecondaryObjects module in “Propagation” mode, which will use nuclei from IdentifyPrimaryObjects as a starting point and expand them to “grow” a cell using information about the staining surrounding those nuclei.

I think the main issue here is that you’re using the same image for both IDPrimary and IDSecondary. As a result both modules produce roughly the same detection. Is this a multichannel image, with one channel for nuclei and one for the cytoplasm? If so, you’ll need to split the channels up using the ColorToGrey module before trying to segment. At present you have NamesAndTypes set to treat the input image as greyscale, which may be combining all the channels rather than treating them separately.

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Hello @DStirling, Thank you so much for your suggestion I made the necessary adjustments and the modules seem to work in identifying primary objects and secondary objects separately. the pipeline created is segmenting the cells that I hoped for, Thank you so much for the help, I appreciate it greatly!!!