Plugin Development - Fluoresence intensity and area - Analyze Particles stack

Hi all,

I’m trying to find a way to analyze an image of fluorescently labeled cells and find the area and average fluoresence intensity of each cell in the image. Has anyone done this before or have any suggestions/ideas for how to do this?


Hi Reberya,

could you provide us some example images? Then it’s much easier for us to help you!



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As @dietzc says, posting a sample image will help. In general, you will probably need to segment your images and then measure them. The Segmentation page describes how to use ImageJ to do that. See also the Segmentation with Fiji workshop.

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Here’s an example of the image I’m trying to analyze. I’ve played around with the segmentation tool a bit and it does a great job of getting the image outlines and transferring them to the original image. The issue is that the measure tool gives me the entire area/intensity vs separating it by objects within the mask.

Thanks for all of your help!

Did you follow the five steps outlined at ?

  1. Preprocess – maybe/probably not necessary for this data since it is very clean.
  2. Threshold (shift+T) – you just need to convert to 8-bit or split channels first to do this. Or use the Color Threshold plugin. (Press L to launch Command Finder for easy access to commands.)
  3. Create Mask
  4. Analyze Particles
  5. Measure (with your original image selected!)

Step 4, Analyze Particles, is really important to split up the foreground from a single monolithic blob into your individual objects. You will end up with all your objects in the ROI Manager afterwards as long as you check the “Add to manager” option. Then for Step 5 you click the ROI Manager’s Measure button to measure each object separately.

See also info about the Macro Recorder to create a macro for reproducing these steps repeatedly, as well as the Batch page for more tips.

@ctrueden Thanks for your help with this particular post as well as my other posts! For future users interested in a function similar to this, the complete macro can be found on this forum page: