FRET-Image acquisition

Before proceeding with our general acquisition parameters several properties of YFP and CFP need to be introduced. First, CFP and YFP photobleach much more rapidly than GFP. After six repetitions of 0.25 second exposures (a total of just 1.5 seconds!) CFP fluorescence is reduced 50%. YFP is more stable, as it takes 6.5 seconds of total exposure to diminish the signal intensity to 50%. The rapid photobleaching of CFP requires that exposure times be kept to a minimum. In practice we have found that in FRET experiments 0.4 second exposure times are optimum. The only exception is for very bright signals where the exposure times can be reduced further. The other practical point is that fluorescence is never examined by eye before acquisition by the camera.