Colocalization problem with imagej analysis

Hi guys,
I’m writing you because I have a doubt about a kind of specific analysis that I make to do on my confocal images.
So I was wondering if you could help me out: I’m a mac with fiji, I have pictures were I need to evaluate the number, but not the percentage, of colocalization between green and red channels. I tried different ways to do this analysis with no success.
Do you know any protocol that I could try?
Thank you in advance for your time and help

Hello @marian, Welcome to the forum.
Could you explain a bit more what you mean by the ‘number’ for colocalisation? Some of methods use co-relations based on pixel intensities that gives you a Pearson value or a Manders co-effiecient. Are you referring to these numbers?
This link will also be helpful -


Hi Praveen,
thank you for replying me.
In my pictures I have to quantify the exact number of yellow puncta per cell. In this case Manders co-efficient gives me a percentage of colocalization for each channel but this is not what I want; I’m interesting to have the exact number. Is there any way to do that?
Thanks a lot

Ok. I would need to see an example image of your red and green channels overlapped to suggest how to do this. Could you upload a TIFF image of one of your images?

Could you please give me your e-mail address so I send you an example?

You can compress and upload the images as a ZIP file here :slight_smile: There is an upload option in your messages.

Original tiff image: download

I cant seem to see the image here. Can you upload it once again?

When I try to upload it shows the message: “Sorry, new users can not upload attachments”.

TIF images are not displayed in most browsers. You can however right-click and then either:

  • Save Link As… or
  • Copy Link Location, followed by File > Import > URL… in ImageJ.

Apparently it did work before. Nevertheless, I increased your account level from New user to Basic user, so you should have permission to upload.


“Yellow puncta” is not a measure of colocalization. Please read here why looking for yellow in a red-green image is not the correct way to quantify this.

In your case, you should measure object-based colocalization, i.e.:


  • measure how many of the red spots are inside or outside the green area, or
  • (if you want more quantitative info about the spatial distribution) measure the distance of every spot to the nearest “green object” using a euclidean distance map (Plugins > Process > Exact Signed Euclidean Distance Transform (3D) in Fiji)

@marian I would suggest what @imagejan mentioned as well. The object based approach is possibly a good way to answer your question.

Hi, have you succeeded with your colocalization analysis?


Hello Vadim Zinchuck,
I really want to contact you to ask about the colocalization analysis.
If you don’t mind could you add my skype: xmaskit