Measure fluorescence intensity

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i am making a paper and i have to measure the fluorescence intensity.
So we have 605 images from neurons which will fluorescence over time (as a respond on calcium). Now i have to choose 5 neurons and make a graphic off them (intensity over time).
with ROI manager i choose 5 areas (neurons) and add them. Then i am stuck. I have to get intensities from all 5 neurons over time so i can make a graph on Excell.
Thank you for responding (excuse broken my english)


Hello @Zahra

Welcome to the ImageJ-Forum.

If you could provide us with some images we could probably help you setting up a solution.


I’m not exactly sure what it is you finally need in the end… But you can play around with the ROI Manager - specifically using the Multi-Measure / Multi-Plot tools… Have a look and see if that provides the data you need.


Hi @Zahra,

If you didn’t already do so, make the images into a stack in time series order.
Then draw your ROIs and add to the ROI Manager as you have already done.
Select all of the ROIs in the Manager.
Then click More >> Multi-Measure as @etarena suggested.
Check the option to measure all slices, and one row per slice.
You can copy the results and paste into Excel.

Hope this helps,


thanx for respondig to you all

when i measure all what do i click in “Set Measurements”? Integrated density?


It depends on what you want to report in the end… have a look at the list of parameters in Set Measurements. The integrated density in theory would be appropriate as it is the essentially the product of the area & mean intensity.


Thanks for posting the image.

For calcium imaging, you want average intensity within the cell, so you should check “Mean Gray Value.” For your graph, you probably want to calculate F/F0 (mean fluorescence at time t divided by the mean at time 0) for each cell.

Notice that your rectangular ROIs include area both inside and outside the cell. This will reduce your apparent intensity and % change, and introduce variability due to varying amounts of background in each ROI.

For the most accurate & reproducible results,

  1. Use the freehand ROI (looks like a bean) to draw exactly around the cell.
  2. Measure the mean intensity of a background area and subtract this value from all mean intensity values before graphing the data.

Also, make sure the cells don’t move outside the ROI during the time series.


we have to measure the fluorescence intensity over time of 5 neurons to check of they illuminate synchronically


Hi @Zahra
I read your question occasionally. Have your problem been solved? I have the similar problem now. Different from your problem, my question is that I just want to measure the fluorescence intensity in s single picture. Can you tell me how to deal with it. Thank you very much


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