Counting colocalized (or not) objects

I’m very pleased to start using the CP. But as expected for a beginner i have had lots of challenges to put my pipeline to work.
The experiment:
I give some small bugs to cells to evaluate the phagocytosis.
The objective: simply know an infection index (cells with intracellular bugs)
The method: the bugs are GFP, the non-phagocytosed bugs are imunostained, the cells can be identified in the phase image. So i have 3 images per set.

I could identify all three objects independently, the problem is how to count per image the cells hosting phagocytosed bugs. To do that I need to count the cells that colocalize the only GFP bugs (not immunostained). The object for immunostaining are a bit bigger than GFP signal.

Please, could you help me.

Provided that you are indeed able to identify the three sets of objects, to count the cells that contain GFP bugs, you can use the RelateObjects module to to establish a parent-child relationship between the bugs (children sub-objects) and the cells (parents). You can then use the FilterObjects module to filter cells by the children bug count, and retain those cells which have a count of 1 or more. In other words, those cells which have more than 1 child co-localize with at least 1 bug.

You can look at the cell/particle counting pipeline on our Examples page for an instance of how this works. Hope this helps!

Thank you. But there is still one problem: using this approach I’ll get the number of cells with Bugs(GFP), but there are two types of bugs: Stained(extracellular) and not stained (intracellular), how to count the cells that colocalize with bugs that are NOT stained (only GFP)?

A similar approach will work here as well: Use RelateObjects to relate all the bugs (parents) and the stained extracellular bugs (children). Then use FilterObjects to remove the bugs which have 0 “children”, i.e., those that co-localize with no stain. This will work provided that the GFP image and the stain image are properly aligned so that GFP/stains co-localize correctly.

Once you have the GFP-only bugs, you can then use these objects in the approach I described previously.



Thank you!