I’ve been having a play around with Ilastik to identify nuclei in images of cells stained with DAPI.
The first set of images I worked with were displayed fine, I could easily distinguish the nuclei from the background.
However, when I attempted the same thing with a set of images from a different experiment the images were displayed so bright that I could not distinguish the nuclei from the much less stained cytoplasm.
Hovering the cursor over regions in which I knew the nuclei were (using the image opened in ImageJ as a guide), showed that there was a pixel intensity difference between the nuclei and the cytoplasm.
Next I tried to correct for illumination, saved the corrected file and loaded into Ilastik and some of the images displayed fine but others were too bright again. Rescaling made things worse, creating a totally black image in most cases.
Is there something really simple I am missing here? Any help would be much appreciated.