I have cell line expressing cerulean and GFP at different time point. I am having difficulty identifying the exact fluorescent protein being expressed since the wavelength overlap for GFP and cerulean.
No you can't, not just using one laser line. Even if you were using two lasers at 405 and 488 and had extremely tight bandpass filters collecting the smallest fraction of light around their emission maximum it would still be quite difficult to cleanly segregate the two. With a single laser line its not possible. Your best bet would be to create a new cell line with more appropriate FP pairs if you wish to segregate between the two. Ideally something in the orange or red region of the spectrum.
I disagree with Howard. You can, and I've done it. It's most possible using filters, as the initial question requests.
You need very tight bandpass filters around 435ex/475em for Cerulean and 490ex/520em for GFP. You cannot do them simultaneously, they must be in sequence when you aquire your data. What makes this possible is that Cerulean doesn't excite up in the 490 area.
Again, sequential.
Thank You Jordan R. Yaron . That helps a lot. Do you happen to know where can i get these filter?
https://www.semrock.com/SetDetails.aspx?id=3348
https://www.semrock.com/SetDetails.aspx?id=2795
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