I need some help with Cell Profiler. I have used this software a few years ago for a basic application, counting cell nuclei stained with DAPI on fluorescence images: it was easy to set up and worked well. I downloaded yesterday the tutorial presenting such an application for the latest software version, it worked perfectly with the test images and all modules looked familiar.
However, the images I have to analyse now are a bit different because they have a “dirty” background: heterogeneous light intensity, artifacts from other focus plans, etc. Here is an example. The objects I want to identify are the cell spheroids inside the capsules.
At first I thought it would be easy to convert the image to grayscale then perform a threshold to have a binary image showing only the spheroids. That’s what I have tried, either directly in Cell Profiler or by using ImageJ first. The final binary image looks as expected:
There are still a few artifacts but I expected the “typical diameter of objects” in the IdentifyPrimaryObjects module to exclude them.
Now here is the problem: IdentifyPrimaryObjects detects a lot of extra invisible objects on the binary image, and doesn’t actually detect the spheroids. I don’t understand how it can detect objects on what I understand to be a “pure” white background after binarization.
I am probably missing/misunderstanding something, but I was really expecting this application to be as straightforward as counting nuclei on a fluorescence image, so I don’t get it.
Thank you very much for your help. Here is the (very simple) pipeline I am using so far: Pipeline gates.cpproj (429.2 KB)