How to use ImageJ to quantify Immunofluorescence image of many cells?

Hello community!
I want to use ImageJ to quantify my immunofluorescence image containing many cells. I want to count the cells in the image and measure the fluorescence intensity of each cell. How can I do that? Any help is much appreciated![Image shows the staining of RAW cells with an antibody recognizing CD45 protein on the cell surface][1]CD45 OLIGO POST STAINING 1 part2-Image Export-08_c1 2.tif (770.2 KB)

HI Anu,

Do you by any chance also have a picture with a nuclear or a membrane counterstain? It will help with identifying your cells before quantification and will avoid bias: you don’t want to measure something which has been selected with what you want to measure…

M.

Hi Matthieu,
Thanks for the response. In this particular example, I don’t. Makes sense what you are saying. How about this image?
M170 oligo destain R1 0min-Image Export-07_c1 2 3.tif (770.2 KB)

Hi Anu,

You need to

  1. Split the image: Image>Color>Split Channels
  1. Select the image with the nuclei and Threshold: Image>Adjust>Threshold. You then obtain a binary file where your nuclei are white. This is where we run into trouble: the resolution of your image is very low and many nuclei are clustered.
  2. You need to find a way to split nuclei that are clustered: try Binary>Watershed to get an OK picture or save this file and export it in Ilastik for better segmentation
  3. Once the nuclei are segmented, use Analyze>Analyze Particles to identify each nucleus as a ROI
  4. Select them all, go to Edit>Selection>Enlarge by a couple of pixels to cover the cytoplasm
  5. Go to the green image, select all your ROI and measure

M.

Thanks so much, Matthieu. This is incredibly useful. Just a couple quick clarifications:

  1. For step 5, should I select in the picture- which is what I did.
  2. For step 6, after I go the green image, How should I select the ROI- should I do it from the ROI manager.
    Thanks once again

Hi Anu,

For step 6, you select the picture then go to the ROI Manager and click on “Show all”. All the ROIs will appear on your image. Click on “Measure” and you’ll get the number you want.

I’m not sure I understand your question in Step 5.

M.

Thanks, Matthieu,
I think it worked. For the image I sent earlier- I got the following values
Count- 2042
Mean- 13.056
IntDen-371.37
RawIntDen- 3422550. Which one of these should I use as the quantification of the signal?

About Step 5- Should I select all the cells from the image or all the values from the ROI manager? Sorry for not being clear.

Thanks a ton!

Hi Anu,

  1. You need to set the measurements you’re interested in. Go to Analyze>Set measurements and select what you need (for you: Area, Mean gray value, Display Label). Choose the number of decimal you need too.
  2. The results you’ve given look like a summary of your measurements, you’re probably interested in the raw data in the table of results.
  3. For step 5: you should see all the ROIs highlighted in yellow around your cells. Select the picture with the green signal, click on the ROI manager and tick “show all”. All your ROIs should be projected on that picture. Click on “measure” in the ROI manager. All your results are in your “result table”, which you can export in Excel for analysis.

M.

Thanks so much for your time and help, Matthieu.