FRET in the time of DPSS

Posted by Tim Feinstein-2 on
URL: http://confocal-microscopy-list.275.s1.nabble.com/Deconvolution-of-Confocal-Images-was-Airy-Units-tp6947651p6979426.html

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Hello all,

We want to spec a four-laser launch for a new live cell system that will handle both CFP/YFP FRET and red/green imaging.   However, I am sad to see that gas lasers are no longer speccable and so the freebie 514 laser line is gone.  We would therefore have to spec a 514 DPSS and forego the far-red line.  

I was wondering whether there is a way to do more (or at least the same) with less.  488 nm excites YFP well enough, so in theory I could image CFP/YFP using a scan head dichroic with cutouts for 442 and 488 nm laser lines.  In my experience 442 nm laser excitation (via TIRF) causes negligible YFP excitation and 488 nm does not excite CFP, so it is possible that I could gain speed by passing everything through a single broad bandpass filter (e.g., 455-550 nm) and alternate excitations.   Assuming that cross-talk is not a problem, the most significant cost would be that I lose a decent chunk of CFP emission to the scan head dichroic, but in return I gain a 641 nm laser.  

Has anyone tried this?  Any feedback on or off-list would be much appreciated.  

Thanks and all the best,


TF  

Timothy Feinstein, PhD
Postdoctoral Fellow
Laboratory for GPCR Biology
Dept. of Pharmacology & Chemical Biology
University of Pittsburgh, School of Medicine
BST W1301, 200 Lothrop St.
Pittsburgh, PA  15261