The CFP was suggested to be excited by the violent 405mm laser and the emission was supposed to be 450mm pacific blue but we found that AmCyan 525mm emission filter worked better. Then, the GFP was excited by our blue 488mm laser and the emission was 530mm FITC. Compensation was set up (FITC to AmCyan is 7.81 and Amcyan to FITC is 6.3). Theoretically, they can be distinguished with the delay time between the two lasers, violent and blue. However, a straight line was still in the dot pot graph, FITC against AmCyan. Is it because GFP and CFP both can be excited the two lasers above? Can you recommend how we should be doing the compensation right? Is it really possible to separate them? I read some other people suggesting using UV 355mm laser and DAPI filter for CFP.
Hi Eric,
I haven't personally used flow cytometry or CFP, but for my work involving different fluorophores, I frequently refer to the online Chroma Spectra Viewer, which I found to be very helpful for sorting out overlapping spectra: https://www.chroma.com/spectra-viewer?fluorochromes=10428,10504
In your example, I would suggest the following parameters, provided you have free control over your excitation/emission (as with a white laser):
CFP excitation: 425 nm
CFP emission: 475 nm
GFP excitation: 490 nm
GFP emission: 510 nm
As you can see, the GFP emission spectrum is almost completely covered by CFP, making it necessary to sort according to different excitation modes.
I've attached the CFP/GFP excitation/emission plot to this post. Dark/bright blue represents CFP/GFP excitation, respectively, and dark/bright red represents CFP/GFP emission, respectively.
Hope this helps,
Oliver
What is the configuration of the fluorophores? Are they fused or do they come in contact with each other via any protein-protein interactions with fusion partners? The reason I ask is that CFP and YFP are a really good FRET pair (and I expect GFP would at least be somewhat able to act as a FRET acceptor in this case). FRET from CFP excitation is detected in the AmCyan channel (ex405,em525/20); free CFP signal should be detected in the DAPI (ex405,em450/50) channel. Of course, if there is FRET going on, the free CFP signal will be somewhat dampened.
The spectrum Oliver attached shows you where your problem is - the CFP is excited by your 488 laser and will give you a signal in the 530 FITC channel (so this would be somewhat weak). Depending on the overall FI of the probes you might be able to compenate for this but I doubt it. There are a number of flow cytometers where the supplier suggest using the same channel for CFP and GFP - indicating that seperating them is usually not the way forward in flow cytometry.
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