I have a simple GFP fluorescent image and I want to count the cell number using CellProfiler. I simply added one module (IdentifyPrimaryObject) to the pipeline but obviously the number of identified objects are not correct. CellProfilers turned 573 identified objects, however, looking by eye, it looks much less cell number. The problem is that CellProfiler detect some clusters and counts many cell in the cluster, although there is not any cell. Anybody could give me any idea how can I adjust the parameters in the “IdentifyPrimaryObject” module to count the cell more accurately. Attached are my GFP image, my parameters in the module and the CellProfiler QA screenshot.