Hi forum, first time posting. I do apologize as I know this topic has been done to death, but I’m still struggling to generate a good pipeline.
Sample image and/or code
JC_Pipeline_Working.cpproj (1.0 MB)
I am plating neurons which all have a green nuclei, and ~1:100 also have red cytoplasm, so that individual neurons can be identified and several features can be extracted. Each well will have 12 stitched images (above I uploaded a single field of view as an example).
I’ve currently put together a pipeline to get the coarse measurement of “area covered by neurites” (attached above), however ideally I would also like to get information about: neurite length, number of branching points in the neurite, and how many trunks extend from each cell body. I think this is the bread and butter of the Measure___Skeleton modules, but so far I have not had much luck with them.
I’ve tried to use “Measure Secondary Objects” by propagation from the ‘soma’ object I identify in the picture, but I find that it often misattributes neurites from one cell to another cell. Similarly, when I try the MeasureObjectSkeleton module on a skeletonized version of the neurite objects, it often calls any crossing point a branch point. My question is: is getting reliable counts of these metrics even doable from images like the one I posted? i.e. should I just keep playing with the parameters, or should I try things on the biology side to make this analysis more robust. Thanks for any advice, I appreciate it!