Counting Glowing Cells: False positives and negatives?

Hello Everyone,

I’m trying to count cells before they are stained with fluorescence and after they are stained (for research). Before they are stained with fluorescence , they all look blue and are nicely shaped. After they are stained, some glow green while others do not. I need to count the number of cells that glow green.

So, what I did was I identified the primary objects of the blue cells, and then I added certain calculations to the green glowing cells. I added measure object intensity, filter, etc.
The problem is that there are scenarios when I see a glowing cell, and it is clearly glowing, yet the computer tells me this black dot somewhere is glowing and NOT the bright glowing cell I see. I don’t know why it does this and I don’t know how it adjust the parameters. How do I get the computer to actually count the glowing cells?

I’ve attached an example and the pipeline I’m using.

Any help would be greatly appreciated!
Pipeline_Count.cp (10.6 KB)


This does indeed seem odd, but I can’t diagnose the issue until I also see the original DAPI and p53 images that you used. Could you upload these files please?