Toluidine Blue in rat heart

Hi all,

I have been attempting to use Cellprofiler to analyse Toluidine Blue stained rat heart for mast cells. I would like to create an index measurement of the number of mast cells (which stain purple) to the total area of muscle tissue (blue). There is some illumination variation across the images. I have made multiple attempts but just can’t seem to ID the muscle area, even with incorporating an illumination correction. The pipeline is attached which is still not giving good results. I am happy to count the very infrequent mast cells by eye but it would be great to have that automated.

Any suggestions or assistance would be fantastic.
The pipeline, original colour RGB TIFF, and the greyscale RGB images (split by imageJ) are attached.


Mast cell restain III LV.cpproj (766 KB)


Please see my attached pipeline. I took your pipeline and modified it, so please note that some of the modules are disabled. I used the color image, since it’s better to simply deconvolve the colors directly from the original source (color).

There are a couple Enhance modules which do attempt to ‘clean up’ the image, but the may need to be tweaked, or even might best be removed. But for this image they do seem to help.

Hope that helps!
Mast cell restain III LV_DLogan.cppipe (11.2 KB)