I am running immunofluorscence staining on human sections. The plan is to measure mean fluorescence intensity in 2D images. In previous studies I used to use imageJ to measure the mean value (open image—>analyze—>measure). Now as I need to measure the intensity in many images, I am trying to develop an automated method in image J to threshold and measure the intensity. I am still not experienced with writing macros in imageJ, however, I have recorded the below simple macro where I first apply “color threshold” then I measure the intensity in single images.
I have few questions in this regards
1- Is applying threshold suitable method to exclude the the black physical holes in the images?
2- Is measuring mean value the best way to get feedback about the fluorescence intensity (thereby the protein levels) in the region of interest?
3- When I want to threshold an image, I usually go to (image–adjust–color threshold), then I select a thresholding method “f.x Huang”. Is that all what I need to do or do I need afterwards to click “select” in order to apply the selected thresholding method to the opened image?
4- As my study include several groups (control vs disease), how can I set the same threshold to all images?
5- How can I threshold and measure the mean value in batch of images automatically without the need to manually open single images one by one, which is very time consuming?.