1. Click on the Individual Nucleus entry in the list and select the Segmentation Method for the class (Global Thresholding).
2. The segmentation parameters (Smoothing, Sharpen, etc.) are displayed below the list.
2. In the Smoothing section, select Gauss from the dropdown list and set the parameter Sigma to 1.5.
3. Click on the blue-stained cell nuclei in the image.
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. In the Separate section, select the Watersheds entry from the dropdown list and set the number to 17.
6. Clear separation lines are now visible between the cell nuclei.
   
6. Click on the Individual Signal entry in the list.
7. The segmentation parameters are displayed below the list.
7. In the Smoothing section select Gauss from the dropdown list and set the parameter Sigma to 1.5.
8. Click in the image on the green-stained signals.
9. The detected signals are overlaid in green. The threshold values are displayed in the Threshold section in the Low/High input fields.
9. Click on the areas of the green signals that have not yet been detected until these have been completely overlaid.
10. Set Fill Holes to On.
11. This fills any holes in the detected signals.
11. In the Separate section, select the Watersheds entry from the dropdown list and set the number to 17.
12. Clear separation lines are now visible between the signals.
    
12. Click Next.