Quantify the whole cell fluorescence intensity (or the intensity of cytosol)

Hi there,

I am trying to measure the whole cell fluorescence intensity using DAPI channel defined ROI.

This is what I did:

  1. Define the ROI using “Analyze Particles” function in the DAPI (nucleus) channel.

  2. I used this defined ROI to read the fluorescence intensity in other channels. So of course, these other signals should be all within the nucleus region.

Q: So now I want to have the intensity for the non-nucleus region but still have the defined cell numbering (see the pic above). I have tried the segmentation function, or binary then erode the boundary a little bit, but both are not very ideal. I am wondering if anyone has a better idea to do this? Real Appreciate!

The original file attached with the zip file here (https://www.dropbox.com/s/v3feu7bk0kucfil/Image%20108.czi?dl=0), pls help to have a look, thanks!

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Not sure if I understood you correctly but here is what i can suggest.
1.Threshold your DAPI channel so that you only have the nucleus highlighted.
2. Use the ‘Image Calculator’ tool to subtract this thresholded-DAPI-channe from the images from other channels,which will give you image ‘A’ that has only cytosolic intensities.
3. Do a threshold on image ‘A’ (without creating a binary image) and run ‘Analyse Particles’ and check for the mean intensities. This would give you the mean intensities of your cytosolic regions alone.


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Hi Praveen,

Thanks sooo much for reply!!

I tried this out and it seems to be a promising way.

This is what I did:

  1. Select the channel in DAPI.

  2. Superimpose the highlighted DAPI ROI manager into other channel. You may could see the cytosolic fraction in this pic.

  3. Subtract the nucleus fraction out so that end up with the pic of this.

  4. Do the threshold and Measure the intensity (overall).

Now, so far so good. But when I tried to use the ROI manager to select the subtracted pic and try to measure each individual cell’s cytosolic intensity. But I realized that I lost the previous numbering when I did the DAPI channel ROI selection.
By this, I mean ideally I want to get a series of measurement corresponding to each individual cell’s nucleus intensity (which could be obtained from pic step 2) and the cytosolic intensity. But I lost them after the operation.

Looking forward for your help!


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Keep a copy of the image 2 which have the selected ROIs. You should also have all these ROIs in the ROI manager. Now choose image 4 and click on ‘Show all’. This will restore your ROIs from image 2 to image 4.
Good luck,

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I realize it has been 4 years but I thought, I would give it a shot anyway.

I am using this similar method of segmentation, however I am having troubles measuring the intensity of protein in cytoplasm of each individual cell. Would you happen to have a step by step guide for this as well? Thank you in advance!