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-tp2576265p2577731.html

We use a Nikon spectral system.  Their detector array is photomultiplier-based so it is quite sensitive for a spectral system.  We use YFP and GFP and it gives very good results.

Craig Brideau
Hotchkiss Brain Institute
University of Calgary, Clinical Neuroscience
Calgary, AB
Canada

On Thu, Apr 2, 2009 at 3:06 PM, Carl Boswell <[hidden email]> wrote:

As a general note, my limited experience with spectral imaging is that unless you have a strong signal, the image quality of the unmixed spectra can be problematic at best.  Of course, it could be the system operator.  If you demo any system, make sure you use one of your own preps, not a commercial slide.

C

Carl A. Boswell, Ph.D.
Molecular and Cellular Biology
University of Arizona
520-954-7053
FAX [phone]
----- Original Message ----- From: "Kurt Thorn" <[hidden email]>
To: <[hidden email]>
Sent: Thursday, April 02, 2009 10:13 AM
Subject: Re: spectral versus well filtered in plants

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

--
Kurt Thorn, PhD
Director, Nikon Imaging Center
University of California San Francisco

UCSF MC 2140
Genentech Hall Room S252
600 16th St.
San Francisco, CA 94158-2517

http://nic.ucsf.edu
phone [phone]
fax   [phone]