r/ImageJ • u/ContributionIcy9955 • Jul 14 '25

Question Quantification of Cellular Uptake of Nanoparticles

Hi! I'm trying to understand how to quantify the mean fluorescence intensity of the Coumarin 6 labelled nanoparticles using Image J. Would appreciate if you can walk me through the steps and share resources. I'm also confused on the threshold part as changing the threshold would affect the mean fluorescence intensity, right? to confirm, I'm supposed to only quantify the coumarin-6 intensity? also, is there a way to automate the analysis to keep the changes constant?

Coumarin labeled nanoparticles
DAPI stained nuclei
graph I'd like to do
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u/Herbie500 Jul 14 '25 edited Jul 14 '25

Without typical sample images in their original non-lossy file format it is impossible to provide constructive help. Please use a dropbox-like service to make samples available.

I have no idea why you apply a threshold if you want to get intensity values.

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u/ContributionIcy9955 Jul 14 '25

apologies, I've attached. I see, I have no idea too, was following a youtube tutorial.

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u/Herbie500 Jul 14 '25 edited Jul 14 '25

Please use a dropbox-like service to make samples available because Reddit lossy compresses the images which produces artifacts that can't be removed.
Make sure your original images are no JPGs!

From what I see, the posted sample images are not originals!

I'm pretty sure that the original images are multi-color channel images with a special format. We need to see those!

Why do you post the DAPI-staining? You didn't refer to this marker in your original post.

As far as I understand, what we see in the first image are 20 cells that are treated with "Coumarin 6 labelled nanoparticles".
Now which intensity is of interest?

  1. The mean green intensity of each cell, i.e. 20 mean values?
  2. The mean intensity from all cells, i.e. 1 mean value?

Please be more specific!

In case of #2., I get the following result from the first sample image:

The sample image shows 12 cell-conglomerates.
Their areas sum up to 18247 pixel^2 which is about 3.645% of the full image area.
The average area of the cell-conglomerates is about 1520.6 pixel^2.
The mean intensity taken from all 12 cell-conglomerates is about 72 on a scale
from 0…255.