How to identify secondary object properly?

I am using human N-C translocation to identify the protein amount inside nucleus and in the cytoplasm (ratio to be more exact).
I am having problem on which the IdentifySecondaryObject will also include the primary object (nucleus) even there is no protein inside, which turns out that my secondary object has the same amount with primary object, but my sample is supposed to have some nuclei expressing the protein and some dont.
Does anyone know how to counter this problem? Or should I measure it using the colocalization pipeline…?

Hi @Olivia_Oh,

Have you tried using a IdentifyTertiaryObjects module to create a Cytoplasm object that does not contain the pixels identified within the nucleus?

There are more details about using this module available in our Translocation tutorial, highlighted in this CellProfiler introductory workshop and available on the CellProfiler Github page.

I hope this helps!

Dear Pearl,

Hi, I think I got it right already! Thank you :slight_smile: