1. You have created a new image analysis setting with the Segment region classes independently method and opened the Image Analysis Wizard, see Creating a New Image Analysis Setting.

1. In the Classes step, click on Classes1 in the list and enter Nuclei in the Name input field.
2. Select a blue color from the dropdown list in the Color section.
3. Click on the Class 2 entry in the list and enter Individual Nucleus in the Name input field.
4. Click on the blue channel icon in the Channel section.
5. For Object Color, click Fixed and select the blue color from the dropdown list.
   
6. You have now setup a class pair for the nuclei.
6. Click Add Subclass.
7. Click on Classes 3 in the list and enter Signals in the Name input field.
8. Select a green color from the dropdown list in the Color section
9. Click on the Class 4 entry in the list and enter Individual Signal in the Name input field.
10. Click on the green channel icon in the Channel section.
11. For Object Color, click Fixed and select the green color from the dropdown list.
    
12. You have now setup a subclass for the signals inside the individual nucleus class (parent class).
12. Click Next.