My pipeline is looking better and better each time I come here for help, which is why I’m coming back! I have attached a picture that exemplifies a few oddities, and I don’t know if I should stop being a perfectionist or if there is still room for improvement. Here are two points I am concerned about looking at this image:
The two rounded cells in the top left quarter (and even the two cells in the upper right corner) looks to me like they have a very sharp boundary, but the declumping systematically fails to identify them as distinct objects. If I play around with smoothing filters and local maxima, I can get it to separate, but then some random unforeseen segmentation occurs in what seems to be a homogeneous object appear…
I agree they look distinct, but no segmentation is going to be perfect- those cells seem to be much smaller in area than the others, which is also going to lead to issues. My rule of thumb for optimizing is to tweak the settings until I have roughly as many objects split wrongly in half as lumped wrongly together- at that point, you just have to declare good enough.
On another note, I have set the thresholding minimum at 0.1 but when you look at the objects, the outlines are encapsulating regions at the edges that are well below that minimum. Is it possible to achieve higher resolution (boundary at the edge of a cell), or is this the best I’m looking at? Such results may skew fluorescent intensities of individual objects.
The resolution should be pixel thin, so either a) there’s still a problem with your “Fill holes” settings (do they go away when it’s set to “None”?) b) those pixels ARE above 0.1 (some of them I notice are next to some pretty bright cells, so the halo-ing that naturally happens in that case could lead them to be- try hovering over some of the pixels you disagree with it calling, what is the brightness reported at the bottom of the window?) or c) it’s a bug. Check a) and b) first, and if it’s neither and you suspect it may be c then upload your current pipeline and a sample image that’s displaying the behavior and we’ll check it out for you.