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:
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
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!