Average Neurite Length per cell/ Longest Neurite Branch length

Longest Branch pipeline .cppipe (13.5 KB)

Hello,

I am currently using this pipeline to analyze adult NSC morphology/cytoskeleton structure. I’ve worked on creating mask images in hopes of obtaining a measurement for average neurite length of all neurites per cell or each individual neurite length or a longest neurite branch length per cell. My experimental condition is a dosage response curve. The increasing concentration is exhibiting less neurite branching, disruption of normal cytoskeleton extensions, and overall what seems to be a smaller measurement for all neurite length per cell.

I am also having difficulty with CellProfiler correctly identifying the nuclei of each cell. I will attach an image below to show my issue especially when two neurites from the same cell branch off from one another at junction and then come together.

Currently, the program seems to be more accurate at measuring individual cells, but I would love to be able to run multiple images through a pipeline that consisted of fields of view with multiple cells.

I’m sorry, I’m having a bit of trouble understanding what exactly the issue or issues are here. Are there two issues, one involving detecting the branching and one involving nuclear segmentation? For either one, we’d probably need sample images in addition to the pipeline you provided to help diagnose the issue and tweak the pipeline; if you can explain a bit more clearly what you think it’s doing wrong and what you wish it were doing instead that’ll also help quite a bit. Thanks!

Hi Beth,

Thank you for your response, and I’m sorry for your misunderstanding.

I hope to be able to detect all individual branches from one particular cell in a field of view. The images I am currently running through my created pipeline have between 40-50 cells on each image.

I was able to crop out individual cells and then run those cropped images through the pipeline, but for time’s concern, I would rather be able to run the original images to obtain a measurement of average neurite branches per cell

The MeasureNeurons module is not very specific about determining the length and location of a vertices for my experiment.

For this original image, it is easy for me to crop out the cells that have migrated away on the left side. Yet, I would rather be able to import the file into my pipeline, for it to measure all vertices branding from each nuclei, and give me an average neurite branch per cell for the whole image.

The MeasureNeurons module is not very specific about determining the length and location of a vertices for my experiment.
I’m not sure what you mean by specific here- do you mean accurate?

I’m not sure you uploaded the right pipeline, because it didn’t actually have MeasureNeurons at all nor any measurement exporting step. In any case, by correcting the intensity gradient and enhancing neurites before skeletonizing it looks like MeasureNeurons actually functions reasonably well on your images, and if you want a per-image average measurement all you have to do is turn on that option in ExportToSpreadheet. The attached pipeline should do basically what you want it to (though you’ll have to add back your display and save steps). If it doesn’t, please explain what isn’t behaving as expected.

Longest Branch pipeline_edited.cppipe (13.5 KB)

Hi Beth,

This new pipeline looks promising, thank you!

My question now is in order to obtain a per-image average measurement, would the neurite branch lengths be found under the length column in the edges output excel spreadsheet?

Thanks again.

No, it’ll be in the Image spreadsheet, under Mean_Nuclei_BranchLength (or something like that).