All true.  We managed to separate CFP, YFP, and GFP plus chlorophyll and
cell walls (blue autofluorescence from 405 excitation) in one sample on a
spectral system (SP2).  It was a bit of an effort, but is possible.  If you
add bi-directional scanning it becomes easier, too.  I can't imagine doing
this without being able to adjust the emission wavebands collected.
cheers,
Rosemary

Rosemary White
CSIRO Plant Industry
GPO Box 1600
Canberra, ACT 2601
Australia

ph 61 2 6246 5475
fx 61 2 6246 5334


On 3/04/09 4:13 AM, "Kurt Thorn" <[log in to unmask]> wrote:

> You won't be able to separate GFP and YFP reliably without a spectral
> system; the emission wavelengths aren't separated by enough to do this
> with conventional filters.
> 
> There are other fluorescent protein combinations you could consider
> which might be better behaved, such as one of the new BFPs or a
> UV-excited GFP like Sapphire which would let you do 4 proteins with
> better separation between channels.  I think you can also multiplex CFP,
> GFP, mkOrange, and a far red FP like TagFP635 or tHcRed.  You could
> probably add BFP or Sapphire to that mix to do 5 proteins, although you
> will probably start getting some crosstalk you need to deal with.
> 
> Kurt
> 
> 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
>> 
>