Quantify fluorescence intensity to analysis mitochondria activity



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!



Sorry for the delay in response… I’m not sure I’m the best one to help here. But I can tell you these things:

That is more-or-less correct. If you want more information Segmentation, just use the following links:

I would think that normalizing your signal measurements for area would be ideal here.

As long as you acquired all images using the same system and acquisition settings, the background should be consistent from image-to-image. If you are worried about this… you could collect that information to be sure.

It is defined for ImageJ as “the sum of the values of the pixels in the image or selection … equivalent to the product of Area and Mean Gray Value” in the ImageJ User Guide, in the Set Measurements section.

I hope this at least helps a bit.

eta :slight_smile: