Mitochondria Fragmentation

Hallo everyone!

I need to check mitochondria fragmentation via CellProfiler (version 4.0.6) for my microscope image analysis (using Mito tracker deep red and Hoechst).
I create pipeline for this purpose (attached), however when I run the pipeline it does not seems that it find all puncta.
In the pipeline, it can detect Mitochondria which is introduced as tertiary object correctly, but in order to find puncta it does not apply to all (attached).
At the moment, I am struggling to solve this problem but since I have no experience using this software, it would be great help if any one have an idea and can help me please.
Looking for any suggestions and support.

Mitochondria.pdf (36.0 KB) puncta.pdf (115.5 KB) MitoTracker_puncta.cpproj (624.9 KB)

You images have too low spatial resolution to visualize mitochondria let alone fragments. You will find it easier if you use higher magnification, resolution, and smaller pixels. You’ll need more than a single pixel per fragment. At very least you can try to deconvolve the images to enhance the details. A spot filter might help to extract the fragments.
Good images yield good results.

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