Quantifying Microtubule Distribution

Image Analysis
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#1

Hello everyone!

I am looking for a way to measure microtubule distribution in cells for comparison with cells in other treatment groups.
I have tried using FIJI to pre-process my images (Subtracting background, thresholding, Difference of Gaussian) and running plugins such as ridge detection to find the filaments, but I can’t seem to find a method that will allow for good quantification or replication.
My thought process was to segment the cells, capture the filaments, and find a way to calculate the area of the filaments that lie within the segmented cells, but I have not had much luck with this.

I’d greatly appreciate any suggestions for methods of quantification, or information about other programs (CellProfiler maybe?) or workflows/pipelines that could accomplish this.

Thank you!

Attached below are two microscopy pictures that I want to compare.

Cells_A.tiff (3.7 MB) Cells_B.tiff (3.7 MB)

#2

Hi,

I would suggest a tandem approach using Ilasik and Cellprofiler. Ilastik is very good for segmentation and finding the filaments will be much, much better. And then that segmentation output can be imported into Cellprofiler as a mask to perform measurements (or ImageJ if you want). Cellprofiler can segment into cells and make per cell measurements also, although the accurate placement of boundaries of cells is always a challenge and won’t be perfect for all cells.

Good luck!

#3

We have a video tutorial outlining using ilastik and CellProfiler here: https://www.youtube.com/watch?v=89XPqczqhvU

and there is a written tutorial on GitHub: https://github.com/CellProfiler/tutorials/tree/master/PixelBasedClassification

Good luck!