Hi,
I have generated a transgenic strain expressing both CFP and GFP tagging two different proteins. However, I am a little confused about the emission spectrum overlap between the two fluorescent tags. I basically aim to look at the levels of GFP after overexpression of CFP tagged protein. My concern is that if GFP and CFP emission spectra overlap, then I might see false increase in expression of GFP where it is actually CFP emission that is adding to GFP intensity. Does anyone have a similar experience and could help me in resolving this using confocal microscope?
Thank you
Hi Joan
It depends on the type of confocal microscope that you have, the filter sets (if applicable) that are available on the confocal, and if it can performing linear unmixing. That being said, if you have a strain that ONLY expresses CFP and one that ONLY expresses GFP then you can use those as controls to make sure your settings are specific.
Please reply with the specifics of your equipment before I can comment further.
Steve V
Hi Steve,
Thanks for your response. Our confocal microscope model is Leica TCS SP5. Yes, I do have transgenic strains expressing only CFP or GFP. Those should be good controls for any levels I measure.
Hi Joan,
I had to ask our microscopy core director for his opinion, because I haven’t used the Leica confocal. His reply:
"You’re right on track. The first step would be to set up some very narrow collection windows that don’t overlap. 450-475 for CFP and 510-550 for GFP. Leica SP2, SP5, and SP8 have freely selectable detection wavelengths just like 710, 780, 880s. If they can mix the CFP and GFP only samples together and image them in the same field of view, this will let them know if there is any cross talk. The CFP signal should be pretty clean, the question is if there is some CFP in the GFP channel.
If there is bleed through, spectral unmixing can clean it up. SP5s can do spectral unmixing, but I don’t know if it’s an extra package that you have to buy and what sort of controls you need."
I hope that’s clear and gives you a starting point, but in the end it’s an empirical determination with your particular samples that will solve the problem. Good luck!
Steve