sentence: Correction of FRET measurements for spectral bleedthrough and cross excitation was calculated on a pixel-by-pixel basis for the entire image by estimating net FRET ( nF ) as follows : nF = IFRET − ( IYFP × a ) − ( ICFP × b ) , where IFRET is sensitized YFP emission ( excitation 430 nm , emission 545 nm ) and IYFP and ICFP are YFP emission ( 545 nm ) upon excitation at 480 nm and CFP emission ( 480 nm ) upon excitation at 430 nm , respectively .