Correction of object intensity?

Dear all,

I am still new with Cell Profiler.
Before asking the question, lemme explain a bit about my purpose of using Cell Profiler.
So, I created a bicistronic plasmid like this construct below:
Promoter --> GFP --> IRES --> GFP and transient transfect it to HeLa cells.
And I want to compare the intensity ratio of GFP and RFP. In theory, if everything goes well, both GFP and RFP would have similar intensity making its ratio close to 1.

Below is my pipelinecontrol u6.cpproj (565.7 KB)

I use RFP image as input to measure the primary object, below is the example:
input%20image

And use the outline from that primary object, I measure the intensity of both RFP and GFP in the calculated objects. Below is the GFP overlay image
overlay

If I look at by eyes, all the intensity of RFP and GFP are roughly similar, however, for some reason, when I calculate the GFP/RFP ratio by dividing the GFP integrated intensity by RFP integrated intensity it generated a highly variant data. Look at U6 for example.Control_v2|500x500

So, I think there is a lot of noise that also included in the analysis. So, my question is, is there a mechanism to correct/filter the object intensity so the output measurement is from 0-1 ?

Or do you have a better pipeline for me to do this type of experiment? Any advice and suggestion is very much appreciated.

Sorry for the long post, I hope I don’t bore you all.

Thank you so much!

Do you mean in the eye piece of the microscope or on the image ?
If you look at 2 images by eye that have different intensities, they might look similar just because the display range is not the same : the display is adjusted for each image individually so that the darkest pixel of an image is displayed as black and the brightest as white, and other pixels have a displayed gray value in between following a linear profile.

To be sure that 2 images use the same display range you can put them in a stack in ImageJ for instance, from there you can tell by eye if one is brighter/darker.
If you dont like the stack option you can also manually do Adjust Brightness/Contrast>Set.. and use the same display range for each image.

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