1. You have set up the image analysis setting with the method ZOI (Zones of Influence) and have opened the Image Analysis Wizard, see Creating a New Image Analysis Setting

1. In the Classes step, select Ring or Ring Element and click Add Subclass to extend the predefined set of classes with a subclass of the Ring Element.
2. Another class below the Ring Element is added.
2. Give this class a meaningful name, e.g. Mitochondria/Mitochondrion.
3. The cell nuclei (primary objects) are stained with AF568 (red channel), therefore you need to select this channel to segment the cell nuclei. Select Primary Object and for Channel select AF568.
   
4. Click on Mitochondrion and select AF488 in the Channel field.
5. The mitochondria are stained with AF488, therefore you need to use this channel for image segmentation.