1. Select the DAPI Individual Nuclei entry in the list and select the Segmentation Method for the class (Global Thresholding).
2. The parameters for the class are displayed.
2. In the Threshold section, set the Tolerance parameter to 1%.
3. Click in the image on the blue-stained cell nuclei.
4. The detected nuclei are overlaid in blue. The threshold values are displayed in the Threshold section in the Low/High input fields.
   
4. Click on the areas of the blue cell nuclei that have not yet been detected until these have been completely overlaid.
5. Set Fill Holes to On.
6. This fills any holes in the detected cell nuclei.
6. In the Separate section, select the Watersheds entry from the dropdown list and set the number to 3.
7. Clear separation lines are now visible between the cell nuclei.
   
7. Click Next.