Re: spectral versus well filtered in plants

Posted by Craig Brideau on
URL: http://confocal-microscopy-list.275.s1.nabble.com/spectral-versus-well-filtered-in-plants-tp2576265p2578304.html

Spectral unmixing software is also invaluable.  We have software in our lab that can handle data from the Nikon system and perform spectral unmixing on it.  It's open source too: ImageTrak

Craig

On Thu, Apr 2, 2009 at 4:40 PM, Rosemary White <[hidden email]> wrote:

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" <[hidden email]> 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
>>
>