Dear Christian

To add to other comments. When using CFP and YFP in plant materials, it 
can sometimes be better to use the 458nm laser line in preference to the 
405 nm line. Despite the increased excitation of YFP from the 458, the 
458 nm line helps to minimise autofluorescence in the plant material 
that can mask low levels of CFP expression.  Still not perfect, but this 
has helped when using Chameleon and other CFP experiments.

When spectral profiling CFP and YFP with autofluorescence, you may not 
need to excite the YFP with the 514 nm line if the expression levels are 
similar or greater than that of CFP. This will help balance the 
intensities so that one probe does not dominate the spectral profiles.

Good luck

Peter

Christian wrote:
>
> Recently a new faculty member has introduced new constructs of both 
> YFP and CFP for localization in plant cells, mostly tobacco and 
> arabidopsis.  Our current system, a FluoView 500 is not set up for 
> this work, so I've been asked to investigate new avenues.
>
> In any case, I think we've all seen a spectral system separate GFP, 
> Sytox Green and FITC in the same cell, but for CFP, YFP, GFP, RFP 
> colocalization in plant cells would a spectral system offer more 
> utility than a system with better filter sets and laser lines?
>
> Obviously there is a cost question in relation to utility here.  Also, 
> here in Midwest of the US, we have primarily Zeiss, Olympus and Nikon 
> systems.  If anyone would like comment on those in particular, that'd 
> be great.
>
> I also have a more specific question, with several groups targeting 
> chloroplasts, I should probably just lean towards a spectral system?  
> Finally, do YFP and GFP overlap too greatly in plants (pH 
> difference?!?) to be separated by either system?
>
> If any has some negative feedback, or suggestions, please feel free to 
> email me privately.  I find folks are awfully nice on the list, but 
> when we're talking several hundred thousand dollars, I need brutal 
> honesty.
>
> Thank you.
>
> Christian
>

-- 
Dr Peter O'Toole
Head of Imaging and Cytometry
Technology Facility
Department of Biology (Area 15)
University of York
PO Box 373
YORK
YO10 5YW

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