We are trying to set up CellProfiler for our high-throughput experiments. We take two separate fluorescent images per well, one for Hoechst (stained nuclei) and one for FITC (Annexin). In the end, we want to have the percentage of FITC-positive cells and the mean fluorescence intensity per well. I have built the pipeline to first identify the nuclei with the IdentifyPrimaryObjects module, and expand the nuclei to roughly match the cell body (ExpandOrShrinkObjects). I check if the FITC signal is within the expanded nucleus by overlaying the outlines onto the FITC image. Then, I measure the intensity of the expanded nuclei objects. I display the intensity measurement on the FITC image and based on that, choose a threshold for positive cells manually and use it in the ClassifyObject module.
The problem I have with my pipeline is that I choose the threshold for pos./neg. myself, creating bias. Additionally, the background of our images is different from well to well, so the threshold I choose for the first well may not fit the images for the second well. I have tried to use the IdentifySecondaryObjects module on the FITC pictures and then use the RelateObjects module, but this does not work out well, as the FITC objects are harder to identify.
Also, we would like to measure the mean fluorescence intensity in only the FITC positive cells, but I am not sure how to incorporate that into the pipeline. How can I improve my pipeline?
pipeline_%pos & MFI.cpproj (139.3 KB)