On the FRET Analysis window, choose the Calculate FRET tab.
Open the FRET Experiment image that you want to analyze in an image window.
Drag the image window number icon from the FRET Experiment image window to the FRET Input Image field. An output FRET Results image file name is automatically generated. (The FRET Results image is saved to the disk and an image window containing the saved image is displayed after the calculation is completed).
In the Donor, Acceptor and FRET fields, validate that the correct channels have been assigned to the Donor, Acceptor and FRET channels of the input image.
Validate that the crosstalk factors are reasonable (these factors were calculated when you calculated crosstalk).
Use the Get functions to specify a background value for each of the 3 channel in the same way as during the crosstalk calculation outlined above. These functions will average intensity values in a box (specified in the Box fields) while you drag the cursor around in the image window with the left mouse button held down. Click on the Get button a second time to disable the Get function in order to proceed to defining ROIs for calculating crosstalk.
Once you are satisfied that all parameters are set up correctly, click on Generate FRET Results Image. When this is finished, the image will be opened and you can proceed on to the Analyze Results step.