FRET Introduction (Page 4)
There are two problems that are encountered when using CFP and YFP as FRET partners. First the FRET channel is polluted with fluorescence spilling over from CFP. Even when CFP is expressed alone a certain amount of fluorescence is observed in the FRET channel.
However we quantify the amount of fluorescence in the FRETchannel when CFP is expressed alone and define a spillover factor:
