I am using Cell Profiler to analyze levels of fluorescence intensity in one channel (red) within neurons, which are stained with NeuN in the green channel. To isolate neurons, I am applying the Threshold module to a grayscale image in the green channel, and then I use ‘IdentifyPrimaryObjects’ to outline the NeuN+ neurons. For most of my images, this system works very well. However, for a subset (perhaps 1/4 of them), which appear to be randomly distributed across my image samples, the IdentifyPrimaryObject module doesn’t identify any objects from the thresholded image, though the thresholding looks very similar between the ones that work and don’t work. The objects are within the size range and are well separated from each other, so I cannot understand why this process selectively works. I’m including the images of this identification step. I would appreciate any help understanding why this is not working for all of my images.