Relating Objects in 3D Analysis

Hi there,

I’ve been using the latest release of Cell Profiler to relate speckles to nuclei by using the Identify Primary Objects for Dapi, Secondary for creating the cells and then again Primary for the speckles. I then relate the speckles to the cells using Relate Objects.
Now I would like to do this in 3D, but I understand that the Primary module doesn’t yet exist in for 3D. Is there a way to bypass this and get the measurement a different way?

Thanks a lot for your reply :slight_smile:

You can create 3D objects using the Watershed module for now; eventually IDPrimary will support 3D but it doesn’t just yet. Once you do, RelateObjects should work in 3D- please let us know if you get errors and what they are so we can get to work fixing them!

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Hi Beth, thanks a lot for your reply. Is there an estimation as to when this functionality will be available?

Hi, The version of CP I have (downloaded this week, 3.0.0), says that Watershed (Advanced folder of modules) does not work in 3D, and I find the documentation is correct.

What are my options for identifying objects in 3D.



Hi there,

Please have a look at

where we demonstrate how to use Watershed plane-wise to identify the object in 3D. It’d be only used at the declumping step.


Hi Steve,

We just noticed that error in the documentation ourselves last week- we apologize for the error! It’ll be fixed in 3.0.1. Watershed is indeed what you should use to identify objects in 3D.

Hi Beth and Min,
Thanks for your pointers and help so far.
I wrote the pipeline you suggested, but I have an odd problem.
I get a run error in RescaleIntensity (‘Invalid dimensions for image data’), and I can not open by doubleclicking on the file names for the images from the Images tab. However, when I check ‘No’ for process images as 3D, the modules will run and I can open the images, but I do not get the 9-pane montage that you show in the tutorial.

I can organize the images in Metadata and Groups, but am using 2 of 5 channels

Thanks again,


Hi Steve,

The reason you don’t see the 9-pane montage once you’ve said it’s not 3D is that it’s now treating each slice as one-at-a-time.

Can you give more info on the on the pipeline, your version, etc where you’re seeing this RescaleIntensity error? Uploading the pipeline and enough sample images to make it run could potentially help too.

Hi Beth,

Thanks for the help, let me know if you have any problems with these.


3D watershedprotocol.cpproj (95.0 KB)

Hi Beth,

It doesn’t like the images loaded, tried twice. Preview showed the images, but now just seeing image names. LMK if I can send by email or file transfer if you can’t access them.



You can probably try to zip them and upload to dropbox or google drive and share us the link. Both are free.


Hi Beth and Min,

Finally figured out my problem. I was too casual in my reading of the section on TIFFs and forgot that they can be image stacks as well as single images. So in CellProfiler, I am just adding three ‘images’ for a 3 channel experiment.

Thanks again for the help,