Comparing immunofluorescent images

I am attempting to compare images of slides that are stained with different colors (green, red and blue)
My goal is to compare the green stain in one image to another without the other colors interfering and it to be the entire image and not pixel by pixel.
The images are not the same so they can not be overlapped.

I want to be able to show in a quantitative way that one image has more green or that the green is more intense than the other image.
I have tried turning the image to greyscale but then I can’t compare the green stain.
I have tried running the align module and image math but haven’t gotten the results I’m looking for.
I am new to cellprofiler and would appreciate any help.
Thank you

Hello @ZKay

First I would look at previous questions within this thread as this is a very common question. Second for the community to help you, you need to provide a detailed question and example images (if you can annotate them) and (if you can a starting pipeline).

For a pipeline comparing cells with different staining
1- spit image into separate fluorescent channels
2- if you have multiple images you can import them together
3- threshold to your desired objects / or measure total image


Thank you for your response.
I have tried searching the forum for help but I find myself still stuck.
Here is one of my images:1lactotlr2.tif (8.0 MB)
I am trying to quantify only the green staining of the entire image and not pixel by pixel.
I split the using colortogrey keeping only the green as a color and again turning all 3 colors to gray- thinking I can then calculatemath and subtract the intensity of the two to get only green value.
However I feel like this isn’t the correct way to do it but I’m not sure of another way to figure it out.
would you mind explaining where i find the option to measure total image?
thanks again

What do you mean?
When you split channels, using ColorToGray module, you’ll have this:

Then you can take the OrigGreen (or whatever you named your image), and make measurements. For example, MeasureImageIntensity, which provides you with total image intensity as well as total area.
You can read more about measurements here.
Here is a quick pipeline:
Test_ZKay.cppipe (9.0 KB)

Thank you so much!
This is very helpful.
Do I understand correctly?
The OrigGreen image now represents only the green particles in the image? and if this is true the measureimageintensity of OrigGreen gives me the intensity of the green in the OrigGreen image which is equal to the intensity of the green color of the original image?

Again thank you so much for your help

You are welcome!
Yes, correct!
All of these colors are pseudocolors. What you have in the OrigGreen image is your real signal from the green channel.

Thank you so much.
I appreciate your help.