Possible to Identify Objects Within Tertiary Object

Hey! New user here - been reading up on identifying objects but can’t seem to find what I want, which is to define an area within which I want to identify objects/cells.

Basically I’ve got the pipeline to idenfify a tertiary object (like so:https://blog.cellprofiler.org/2016/12/21/help-what-are-these-three-different-modules-to-identify-objects/) (except there’s just one object, always, not multiple) and what I want is to then identify objects again within that defined area, “cytoplasm”. Like if they were vesicles, or something.

Is there anyway to do this?

Thanks for the help!


In your specific problem, how do you define the “area within which I want to identify objects/cells”. Depending on your problem you might be able to and would want to define it as a primary object (Area) and relate several primary objects (Cells) together. Can you post and example Image?

Yeah, the original poster might want to use the defined region “cytoplasm” as a mask, then go use IdentifyPrimary objects to identify the small objects within just that region. I’m not super familiar with how masking is applied though.

Anne just explained it, and I don’t know what else you would need.

So basically the pipeline would have:

  • IdentifyPrimaryObjects : input = nuclear channel | output = Nuclei
  • IdentifySecondaryObjects : input = cell channel | output = Cells
  • IdentifyTertiaryObjects : input = Nuclei & Cells | output = Cytoplasm
  • MaskImage : input = vesicle channel, masked by objects "cytoplasm | output = MaskedImage
  • IdentifyPrimaryObjects : input = MaskedImage | output = Vesicles

Thanks for the help! Basically I want to use this image (DAPI stain) to idenitfy the whole mass as one “object”,

then use this image to identify a GFP region as a separate object, ,
and then identify the smaller objects in the remaining area between the two objects (the little floating bits outside the main GFP mass) so I can count them.

I’m going to give the masked image method outlined below by Anne and Minh a go and see if that works.

1 Like

thanks for this! I’m going to give it a try

I see.

Here is a very similar idea that was discussed Analysis of diffuse and uneven muscle staining

We always encourage everyone to include example images right at the first post.

Good luck.

Dear Mihn,

This was great, thank you!

I’m trying now to get ExportToSpreadsheet to spit out the number of primaryobjectsidentified in the second round out - however it keeps giving me something like this:
where it lists every single object identified - is there an option to have it just output the # of accepted objects like it does when it runs through the steps of identifying primary objects
for each image set I give it?


Hi there,

The quickest method is just using that exact Excel sheet and do the function COUNTIF onto the column “ImageNumber”, as explained here:

So the number of occurrence of Image number 1,2,3,4… will immediately give you the number of accepted objects on each image.


1 Like


You can also just look at the “Image” spreadsheet- it should have a Count_ObjectName column for each object that tells you how many there are of each object in each image.


Yes I noticed that not long after asking haha - thanks!

1 Like