I was hopeful you could provide assistance with my analysis.
At the bottom of this description, you will find a link to the two images discussed below.
The first image is a cross-section of muscle fibers which were stained for MHC-1 (green). The black fibers are therefore MHC-IIa or IIx containing fibers by process of elimination. Unfortunately, the “background” of the image is also black, which is causing some of the problems, even when conducting pixel classification in ilastik. DAPI was used to identify myonuclei.
The second image is a stain for a protein within the cytosol. The more intensely red the stain, the more of the specific protein being probed for.
Critically, I want to calculate the fiber-type specific contents of this protein, based on the intensity of the stain using CellProfiler. I was hopeful you could provide an effective pipeline to do so as the pipelines available on the site, and custom pipelines I’ve built are not producing trustworthy results:
Here’s the breakdown of what I want to do.
I need to remove background noise and segment/separate type-I fibers (green) from type 2 fibers (black) effectively. I do not need the myonuclei for this analysis, so eliminating them is fine. However, if you can provide a way to keep those in and separate them from the fibers to count them and calculate their size, that would be helpful.
I then need these fibers labeled or classified as their own specific “object”. That is, I need them to be identified as type 1 or type 2 fibers in order to calculate fiber-type specific protein amounts.
I would then like to overlap or align this fiber-type classified image with the second image (red stain) in order to elucidate fiber-type specific amounts of this protein more accurately and efficiently. I am aware this will likely require gray scaling and that multiple modules can be used for various steps, but wanted to ask for your expertise to expedite this as much as possible.
I look forward to your assistance.