we would like to quantify small DAPI-positive spots around the cell nucleus. In order to detect even the very small ones, which are very close to the nucleus, we decided to first identify the nuclei with relatively stringent settings (plus distinguishing clumped objects) and then to crop the original DAPI-image in the shape of these objects. We would then substract the cropped image from the original and use the resulting image to identify the small spots. However, this doesn’t work properly because the pixels along the lines which divide clumped objects are not treated as object pixels and are therefore not contained in the cropped image and ,in turn,tried
are present in the resulting substracted image.
I have tried to get around this problem by expanding the objects, but they don’t expand at the position of the dividing line.
I don’t know whether it would help to unify objects that are touching before the cropping step, but I think the unify module is not available in the recent versions.
I would be very grateful if anyone could help me.