I am trying to analyse the size and intensity of focal adhesions (FAs) in my samples and am having a hard time segmenting individual FAs. Plus my pipeline misses a few of them. In addition my pipeline also picks up actin stress fibres at the edge of the cell elongating the FAs. I would like to avoid this from happening but may be impossible. I was wondering if you could help. I am also posting examples of starting and analysed images to stress improper segmentation and the pipeline used.
Just to give an idea of things I have already tried:
In the identify primary objects module:
I have tried smoothening filter size from 1-20 instead of auto…did not help
I have also tried the same filter size for " minimum allowed distance between local maxima"…did not help
In the enhance supress module:
I have tried using enhance neurites instead of speckles…helped a little bit in segmentation but ended up identifying too many actin tails
Also tried using enhance neurites and enhance speckles one after another and vice versa …did not help.
I do not not know how to manually add segments …would really appreciate your feedback!!
FAidentificationfinal.cp (9.14 KB)