I am learning how to use Image J to calculate fluorescence intensity. I saw the pictures of staining oocytes which show 2 colours (red and green), but I am not sure how they quantify the red and green fluorescence intensity from these pictures.
I guess they did following below steps:
1) In each red or green fluorescence image, they chose 1 oocyte to be ROI
2) Then set functions: Analyze -> Set Measurements -> chose "Area, SD, Min and Max gray value, Integrated intensity, Mean gray value"
3) And then chose "Analyze -> Measure" to calculate red or green fluorescence intensity. After that, a result table appeared, and the red or green fluorescence intensity was "Integrated intensity".
Could anybody please check for me:
- Are these steps right or wrong?
- Is the red or green fluorescence intensity affected by area/size of oocytes? In case I have several oocytes in one image (some oocytes are smaller than others), and I want to calculate the red or green fluorescence intensity for each oocyte, do I need to choose an identical area for oocytes? And after having fluorescence intensity for each oocyte, can I calculate the mean of red or green fluorescence intensities from it?
- Do I need to care about the Mean fluorescence of background?
- What is "Raw integrated intensity"?
I am so confused, so I really appreciate any help from you.
Thank you in advance!