This custom statistical feature calculates the mean translocation ratio of cells in a well. For this, your analysis setting has to use the method ZOI (Zones of Influence) and needs to be set up to detect cell nuclei with a definition of an appropriate width for the ring to cover the cytoplasm near the nucleus.

In the Statistics step, define the feature Translocation Ratio and calculate the mean intensity in the second channel in the nuclei divided by the mean intensity in the same channel in the ring. Therefore, define the following operands and expression:

• Operand a:
• Class: Primary Object
• Feature: Intensity Mean Value of Channel 'EGFP'
• Statistical Operation: Mean
• Operand b:
• Class: Ring Element
• Feature: Intensity Mean Value of Channel 'EGFP'
• Statistical Operation: Mean
• Define Custom Expression: a/b