Hi to every one,
I’m trying to quantify fluorescent intensity in my images to quantify YAP expression in these cells. My problem is if I quantify the total intensity of YAP (red channel) per cell, the mean is diluted by all the black present in the signal inside the cell because YAP is not expressed equally in all the compartments. So, I tried to make a binary mask after a gaussian blurr (1.5px) and a median filter (1 px) and thresholding to catch all the signal from 20 to 255. After this, I make several operations of erode and open trying to have a good area and delete some points produced in the thresholding step.
From this binary image, I created a ROI that I used to delimit the quantification in the original YAP IF image, keeping mainly with the perinuclear area which is my area of interest.
Do you guys think it is a good way to quantify YAP or is it another way to do this? I have attached some photos of the process.