With the help of the forum, I've solved how to measure separate RGB intensities using the same selection. My next question relates to making an actual selection. The purpose of what I am doing is, using skeletal muscle stained for three differing myosin heavy chain isoforms, to quantify fiber type and area based on the relative intensities of each isoform (e.g. if a fiber has 75% red, 20% green, and 5% blue- I would quantify that fiber as a predominantly 'red' phenotype).
My 'issue' is the lack of consistency I am able to produce by manual drawing of the fibers. I have played around with threshold function a bit. However, it does not seem to truly 'get the job done'.
Do you guys have any advice as to how I can more reliably make the area drawing, then use the area drawing to measure size and relative intensities?
Thank you. I am attaching a sample image for reference. An accessory problem is the lack of membrane stain for our samples. However, we are unable to run the samples again.