Sorry I was slow to respond – we had a software release and things have been busy! In any case, it was fine to post in this thread. Have you progressed in the meantime?
I updated the beginning of your pipeline to our newest CP release. I did this because a new thresholding method in IdentifyPrimaryObjects works pretty well here, and it is only available in the CP 2.1 release. Please see the attached project. Here are some notes/tips
(0) Launch the new CP 2.1.0, and load the attached project. In the Images module, drag your images for a site(s) into the window on the right to load them.
(1) I took out the ApplyThreshold modules. They don’t do any background correction per se, and they are unnecessary as far as I can tell since an IDPrimary with proper settings will provide the same functionality.
(2) One channel (at least) needs background correction (Blue). The CorrectIllumination* modules help here. You could apply duplicates for the other channels as well, if needed.
(3) IDPrimary: Your size range was too small for the objects. I am using “Automatic” which defaults to the new “MCT” method. I also removed any lower bounds that you had (0.50 I believe), which are dangerous especially when there is so much background and the hard threshold/lower limit is not usually robust across images/sites.
From here, you could add IDSecondary and all your Measure modules, etc, but I am not quite sure what you mean by vesicles vs. the 3 cells (nuclei?) that are the most obvious objects in these mages. If you need more help, please mark up an image to define what your compartments are in a sample image.
DL_project.cpproj (533 KB)