Identification of primary objects on empty images


#1

Hi all,

I’m trying to identify dead cells in the Cy5 channel. Those are colored on top of the nuclei, therefore I’m using the IdentifyPrimaryObjects module. This works fine if there are dead cells in the image, however, those are very rare, and therefore I’m getting random signals identified (there are no dead cells) on the other images.
After having identified those dead cells, I would like to exclude those objects from the further analysis (preferably automatically for all channels). Is there a way to do this (e.g. get the object ids and exclude them from further analysis)?
Thank you very much for any assistance

Cheers, Anja


#2

Hi Anja,

This works fine if there are dead cells in the image, however, those are very rare, and therefore I’m getting random signals identified (there are no dead cells) on the other images.

You should check your module outputs to be sure, but if as I suspect the images where there are NO dead cells have a very low threshold in IdentifyPrimaryObjects (like 0.01) compared to ones where there really are dead cells (like 0.2), you should be able to set an threshold minimum so that only “real” dead cells are called.

After having identified those dead cells, I would like to exclude those objects from the further analysis (preferably automatically for all channels). Is there a way to do this (e.g. get the object ids and exclude them from further analysis)?

Yes! You can use the FilterObjects or MaskObjects module to exclude the nuclei from dead cells- which one you will choose depends on the details of your pipeline, so I recommend reading the help for both to decide which one is right for you.

Good luck!


#3

Hi Beth,

thank you very much for your reply. I managed to deal with the images with no dead cells by setting the intensity to 0.003 (the max is at about 0.004, so not a strong signal at all…).

I’ve tried using the MaskObjects module before I posted the questions, but the masked cells are still being identified by the IdentifyPrimaryObjects module that aims to identify the nuclei…

deadCells masked

nuclei

I assume there is something wrong in the way I call the MaskObjects module, but not sure what it is… I’ve attached the pipeline. Do you need an example image as well?
testDC.cppipe (29.9 KB)

Cheers,
Anja


#4

Hi Anja,
What you want to do is to mask the nuclei by the deadCells, so you’ll want to put that module after the point where you ID the nuclei and change the settings accordingly. Does that make sense?


#5

Hi Beth,

yes and no. Because when I go on to identify the secondary objects, I would usually use the primary objects as a starting point. But when I mask the primary objects after identification, the mask isn’t included in the identification of the secondary objects. And when I change the input image to the one with the masked objects, it only finds a few or no secondary objects… Where am I going wrong?

dead cells:



#6

So if your use case is what I think that it is (you want to identify areas that are likely to be dead cells, identify nuclei, throw out the nuclei that correspond with your dead cell marker, then use the remaining ones for your downstream analysis), you want to invert the mask in MaskObjects (Set ‘Invert the mask?’ to Yes)- that’ll let you keep only nuclei that DON’T overlap with the dead cell marker->call them something like LiveNuclei. you can then use LiveNuclei as the seed objects for IDSecondary to find your cells.

Was that more clear? If that’s not your use case, please let me know exactly what it is and I’ll try to help you further.


#7

Yes that makes sense, thank you very much!