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We have never been able to get good CFP imaging with 405 nm excitation.  
I think you need a 440 nm diode if you really want to image CFP.  
Alternatively, just switch to Clover / mRuby2 for your FRET experiments; 
that's what I've been encouraging users here to do.

Kurt

On 12/6/2013 9:44 AM, Knecht, David wrote:
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> The multiline argon laser on our Nikon A1R confocal died the other day and we have been considering what to do.  It seems that gas lasers are disappearing and replaced by solid state.  The only reason we have the multiline argon is so that we can do CFP/YFP FRET with the 457 and 514 (or 488) lines of the gas laser.  However, looking at the excitation/emission profiles it appears taht we could do as well or better using the 405 for CFP and 488 for YFP.  We should get slightly less CFP signal with 405 but much less YFP non-FRET signal coming from 457 excitation.  So I would anticipate the ratios to be at least as good or better. Has anyone directly compared these two scenarios?  If 405 works fine for CFP, then we only need to get a solid state 488 which would simplify the repair.  Thanks- Dave
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> David Knecht, Ph.D.
> Professor and Head of Core Microscopy Facility
> Department of Molecular and Cell Biology
> U-3125
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> University of Connecticut
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