1. You have acquired or opened an image of a sample with a signal of the acceptor fluorophore only using the same imaging settings which are later used for imaging the FRET sample.
2. You are in the FRET view.

1. Go to the FRET view options tab and use the controls to define one or more analysis regions in the raw data image.
2. The regions are displayed in a table on the FRET tab.
2. Make sure that Object is activated for each region entry in the table.
3. The regions are defined as objects that should be analyzed.
3. On the Parameters tab, click Acceptor.

1. The acceptor coefficient values are determined for the defined regions and displayed in the Parameters tab as well as the data table below the images.