Intensity Ratio Nuclei Cytoplasm Tool help

Hello,
I’m trying to use (I’m very new to ImageJ) the Intensity Ratio Nuclei Cytoplasm Tool for ImageJ to calculate the ratio between the nucleolus and the nucleus (which should work similarly to nuclei/cytoplasm). I have performed the AgNORs staining on my sections.

I am having troubles with the threshold for the “cytoplasm”(in my case the nucleus) as some "cytoplasms"are not included in the evaluation… Can somebody suggest a good setting under “s”(tab) that could help me include all my “cytoplasms”? Is there an option to see a ratio for every cell or the plugin only gives you a mean value?
I have attached a snap for you to have a look at (*hopefully it worked).

Thank you!

Hi @saravetvet,

the tool has been made for fluorescently stained cells. It expects the background to be dark and the cells to be bright. The control image shows the background in blue, the cytoplasm in grey and the nuclei as outlines. As you can see in your image, the nucleolus are considered background and the background is taken for nucleolus.

Technically you could invert the contrast of your images before running the tool. But I can not tell, if the measured intensities would be meaningful in your case. There is a lot of assumptions that have to hold for this. Linearity, a reasonable homogeneous background, is the ratio comparable between different images and so on. The way the background is determined could be problematic here, since it looks for maxima around minima.

Best regards,
Volker

I forgot to mention that the tool measures average intensities per image, not per cell, to avoid having to segment the cytoplasm.

Thank you for your reply @volker ! I really appreciate your help.

Do you have any suggestion about other ImageJ plugins that I could use or tutorial I could have a look at?

Thanks

Sara