Glad to see a nice pipeline, (although I don’t quite understand the purpose of the series of 4 “Threshold” modules)
Regarding the hole in image 126 (isn’t it a blood vessel?) :
I gave it a few try and feel it’s a bit too hard to use just CellProfiler to automatically differentiate blood vessels with large fibers based on only 1 single channel. If you have other channels that’s more or less specific to tell fibers vs. non-fibers , it may be more feasible.
As you already pointed out, we can try hard to filter the hole/vessel perfectly in 1 particular image, for e.g. image 126. But the filtering mechanism may fail in image 89, where there’s only parts of the vessels.
What I have in mind is:
base on the fact that vessel wall is kinda thick, you can try to use ilastik (or other computer vision methods) to identify just the vessels, then mask them out, then feed Cellprofiler with the masked images to identify the fibers.
However, in such method, “the number of clicks and tweaks and fine-tuning” may be even more than doing manually hand-drawing the regions of exclusion per image, and feed the masked images for automatic fiber identification.
Hope that gives you some ideas.