To calculate crosstalk:

From the softWoRx toolbar, choose Measure | FRET Analysis. Make sure Calculate Crosstalk is the active tab.
Open the Donor and Acceptor images in separate image windows.
Drag the image window number icon from each of the Donor and Acceptor image windows to the Donor and Acceptor window fields in the FRET Analysis tool.
Validate that the Probe Channel for the Donor Control Crosstalk is set to the correct (Donor) channel and the Probe Channel for the Acceptor Control Crosstalk is set to the correct (Acceptor) channel.
Validate that the FRET Channel is set to the correct channel for each control image.
Specify the background for the Probe and FRET channels of each image by using the Get buttons as follows: Click Get and drag the cursor around in the image window with the left mouse button held down. (As you drag the cursor, the Get function averages intensity values in a box, specified in the Box fields). When you are satisfied with the background value, click Get a second time to disable it.
For each image, use the FRET Analysis toolbar to define representative areas (Regions of Interest or ROIs) where FRET would occur if these were Experiment images.
Click Calculate Crosstalk to generate a crosstalk factor for both the Donor and Acceptor control images. Typical Donor Crosstalk factors are 50-70%; typical acceptor crosstalk factors are 15-30%.

The donor and acceptor crosstalk factors will be loaded into the appropriate fields for FRET calculation.