I am new to CellProfiler and I have a problem with automatic object identification during my analysis. My cells are transfected with GFP, nuclear staining with Alexa-568 and DAPI. I am interested to evaluate Alex-568 and DAPI intensity in GFP+ and GFP-negative cells separately. The transfection efficiency for my cells is very very low. Next, I am trying to subtract the background intensity.
I have tried to make several pipelines. Manual object identification is perfectly working but I don’t understand how to identify the GFP positive objects automatically and how to subtract the background intensity in each channel?! Please help me with this. I have attached my pipelines and one image. Please let me know if you need more information.
Thank you very much in advance.