From the softWoRx main menu, choose Measure | Colocalization (ROI) to open the Colocalization Analysis window.
in the Input field, Enter the number of the Image window that you want to analyze.
In the Input Channels lists, select which channels to analyze.
To select specific areas of
the image, use the freehand polygon 
to create
polygons. Then use the copy polygon 
and the
paste polygon 
tools to
copy and paste the polygons through time points or Z sections.
Select a background threshold for each channel by clicking Get and then clicking on a background area of the image. The background is an averaged value within a box of the size specified.
Click the Get button again when you are finished selecting the background.
Click Do It to plot the Colocalization graph and calculate the Coefficient of Correlation value (this value is displayed in the Colocalization Analysis window).