Identify secondary and tertiary objects in the Cell Profiler

Hello everyone,

I am using Cell Profiler software for nuclei counting bu using “identify primary objects” setting. But I have a problem to set up secondary and tertiary objects. I could not set up them for counting other cells in addition to nuclei. I am wondering is there any sources or ways that you can help me? I am extremely grateful to you in advance.

Hi @Nigar,

Could you expand on what you mean by counting “other” cells? An example image might be helpful.

The idea of IdentifySecondaryObjects is to take some Primary objects and “grow” them, typically this would be to find the boundaries of a cell when starting with nuclei. If you’re looking to find a completely different set of objects, another IdentifyPrimaryObjects module might be the solution.

Hope that helps.

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Hi @David,

Thank you very much for your response. It is very helpful.

I am using IdentifyPrimaryObjects for counting nuclei within the whole population. But now I would like to count specific cells within the same population. Their percentage is so low, but still it is worthy to count them. I think I should do IdentifyPrimaryObjects setting again. Do you think it is okay to do two IdentifyPrimaryObjects setting within the same pipeline ? Thank you very much.

There’s nothing wrong with using a second identify module. However, if those rare objects are already picked up by the first Identify module then you may want to use the FilterObjects module instead to isolate the cells of interest.

If you do end up identifying them separately, you can use the CombineObjects module to merge the two sets together before downstream processing if that’s helpful.

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Thank you very much.

Dear @DStirling, sorry to bother you again. I tried to use filter objects module, but unfortunately could not manage to put min and max diameter. Rare cells are bigger than others despite of their less amount. I want to get numbers of nuclei including rare cells, and rare cells alone. As you mentioned, I can combine them in order to get the results. But not sure how to do that. Thank you very much.

In order to filter by size in FilterObjects, you’ll probably need to run the MeasureObjectSizeShape module on the objects before the filter module. This should record the measurements you’d need to exclude based on diameter. Have you tried that?

Thank you very much. I tried it but still have problems related to threshold.

Hi @Nigar,

We’d be happy to take a look at your pipeline to see if we can help. If you share an example image and your project file, we can try to help you troubleshoot your threshold problem and the FilterObjects.

All the best,
Pearl

Dear @Pearl,

Thank you very much for your message. If it is okay, can we have Zoom meeting, so I can show my pipeline and issues ? Thank you very much.

Best wishes,
Nigar