Using the binary image you attached… I did the following steps:
- Threshold the binary image
- Create Selection - I also added it to the ROI Manager
- Skeletonize - I did have to do an inversion before this step to make sure the skeletonization was occurring within the bounds of your cell layer
- and Analyze Skeleton to calculate the longest shortest path (make sure you select that calculation)… this is your center line along the length of your cell layer
For me - this workflow was fine. Try again and let us know.
And then look at this old forum post for finding your values along that line:
That should do it!