Measuring fluorescence intensity



Hi, I have just got my first batch of images and is trying to analyse the density of positive staining I have in a particular region of interest. After reading and searching for various ways, I wonder if it is ok to use integrated density- after I threshold my images. - all my images are taken on the same florescence microscope on the same settings/exposure/magnification- also I was wondering if calibrating all the images is a must for analysis or theoretically all these images would have the same pixel density as they are all taken in the same way?



Hey @fishxy88

What you propose seems fine - integrated density sounds appropriate - but more info would help. You can post an example image (preferably the original) and ROI too here - which would be helpful for us to see, as well as a more detailed description of what you want to measure and why.

Too - just a word of caution on measuring intensity on the same channel you use to set your thresholds (hint: it’s not a good idea):

I also found this helpful exchange on Research Gate which you might find helpful.

eta :slight_smile:


Hi @etarena

Thank you for replying me, allow me to explain what I am currently doing for my data

I am working on mice brain sections stained with a specific marker for pro-inflammatory microglia- I would like to see if there is any difference in the pro-inflammatory microglia activity among different treatment arms around the lesion site

  1. I open all 20 sections of coronal cuttings of a single mice brain
  2. I manually adjust the brightness min/max to get similar levels across all sections
  3. I threshold all using the same values-> the current setting is 40/255
  4. I select 2 ROI with same pixel size (400x400) around the lesion site (one on the left one on the right)
  5. I measure: area, mean, int density, %area
  6. I repeat step 4 and 5 for all remaining brains and I get a mean of the *primary measured outcome (which is what I am confused about) and compared with mean of other brains from different treatment arms

Here are the confusing parts:

  1. I did not calibrate my images as they are all taken with the same microscope at the same magnification by the same software at the same exposure- it was batch processed
  2. I am confused between int density and %area: I have read the definition of each on the imagej user guide

Integrated density: The sum of the values of the pixels in the image or selection. This is equivalent to the product of Area and Mean Gray Value.

Area fraction: For thresholded images is the percentage of pixels in the image or selection that have been highlighted in red

So I was also considering to use area fraction now as it seems to suggest that it represents the percentage of highlighted pixels in my ROI, will the data be of my intended meaning? Because what I want is how much of activity going on in a specific area of same size across all the brains

Here is a dropbox linke to sample of my image (it can’t seem to upload here), already adjusted with my desired brightness - the threshold value that I will be using is 40/255 across all images and I have two ROIs on it.

Thank you so much for your help


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