Maybe you could use mTeal instead CFP and Cerulean instead YFP. This FRET pair is more efficient and it overcomes the problems of the FRET pair CFP-YFP! Good luck :-)

if vitamin C do no disturb your measurement, you could try to add vitC or other scavengers like uric acid, GSH or staff like that to your buffer. 

Thank you Daniel and Emrah for the advises.

We have found mTurquoise2 to be far superior to the other CFP variants in terms of photostability.  We routinely use it for acceptor photobleaching with YFP.  You should also use the minimum amount of 514 necessary to bleach the YFP >90%.

Thank you Jay,

My plasmid has mTurqouise-GL as CFP but still, I have bleach in my CFP.  Furthermore, when I reduce the amount of power laser to do the bleach, it does not bleach well just around 10%. Do you have a protocol of the settings that you use for your acceptor photobleaching experiment? 

Thank you

 Did you make sure that the bleaching occurs due to 514 laser? I use mTurquoise-mCitrine FRET pairs and has not seen photobleaching of CFP on YFP excitation (514 laser) in Zeiss LSM780 even with 40 to 50 Iterations at 100% laser. But 458 nm can definitely increase CFP photobleaching. To reduce CFP bleaching in Acceptor photobleaching, I use 405 nm for CFP.  You could try that if you have 405nm laser line as CFP can be visualized with 405 laser too.

You can make sure if the bleaching is due to YFP excitation by using CFP only sample -> Capture one image with low CFP excitation and exposure followed by 514 nm laser bleaching-> Capture few more CFP images and compare the CFP intensity before and after bleach. Do the CFP before/after calculation for unbleached region also. 

Thank you Bobin, I will have a look to those control.

If you use fixed cells, what kind of mounting medium do you use? I noticed that commercial mounting media inhibit YFP bleaching so you need more time to bleach it. Eventually, you could have partial bleaching of CFP too. I use PBS 20% -glycerol 80% pH 7.4 to obtain rapid and effective YFP bleaching.

Hi chiara,

you are right, I solved the problem using a mountain medium that it is based on Glycerol from Invitrogen, that does not use antibleach reagent.

So far I am getting good results with this new medium. In the future , I will use your medium because is cheaper to prepare.

Thanks