I just started working with your software and so far am very impressed. I’m not even scratching the surface but can still see the power of this tool.
My problems are probably quite basic to everyone here but even still have been frustrating me.
I have two cell populations: GFP+ and GFP- cells. I am only interested in measuring the GFP positive cells therefore I would like to first identify nuclei (using DAPI) then correlate this to GFP expression (secondary objects). Not every nuclei corresponds to a GFP+ cell. However, when i do this the program identifies cells which clearly are not GFP+. Even when the background is completely black it “identifies” and outlines cells. Identifying the GFP+ cells as primary objects and nuclei as secondary doesn’t help as well.
As you can see from my pictures, I have some clumped up cells that are relatively round and more intense in the centre. I would like to be able to distinguish these clumped up cells since I want to do a neighbour analysis. Right now my pipeline just completely ignores the nuclei problem and looks at GFP+ cells. I threshold using Background Adaptive: 1.3 and a Laplacian of Gaussian method to distinguish clumped objects, with minimal success.
My feeling is that using the nuclei would be easier to distinguish clumped cells, however, not every nuclei corresponds to a GFP+ cell.
Please, if anyone could help with my basic albeit trying questions I would be most grateful. Thanks.
ClusterPipeline.cp (5.67 KB)