spectral versus well filtered in plants
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Christian-103 |
spectral versus well filtered in plants
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Kurt Thorn |
Re: spectral versus well filtered in plants
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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 415.514.9709 fax 415.514.4300 |
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Boswell, Carl A - (cboswell) |
Re: spectral versus well filtered in plants
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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 520-621-3709 ----- 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 415.514.9709 > fax 415.514.4300 > |
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Armstrong, Brian |
Re: spectral versus well filtered in plants
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Hello, I think that you would not be happy if you try to separate these
spectra by using filters. You will need a spectral detector. Both Zeiss and Leica make very good spectral Confocal microscopes that will do the job nicely. You may also want to consider the Nuance system from CRI for around $80K US (no commercial interest). Cheers, http://www.cri-inc.com/products/nuance.asp Brian Armstrong PhD Manager, Light Microscopy Core Beckman Research Institute 1450 East Duarte Rd Duarte, CA 91010 626-256-4673 x62872 http://www.cityofhope.org/SharedResources/LightMicroscopy/LightMicroHome .htm -----Original Message----- From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Carl Boswell Sent: Thursday, April 02, 2009 2:07 PM To: [hidden email] Subject: Re: spectral versus well filtered in plants 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 520-621-3709 ----- 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 415.514.9709 > fax 415.514.4300 > --------------------------------------------------------------------- SECURITY/CONFIDENTIALITY WARNING: This message and any attachments are intended solely for the individual or entity to which they are addressed. This communication may contain information that is privileged, confidential, or exempt from disclosure under applicable law (e.g., personal health information, research data, financial information). Because this e-mail has been sent without encryption, individuals other than the intended recipient may be able to view the information, forward it to others or tamper with the information without the knowledge or consent of the sender. If you are not the intended recipient, or the employee or person responsible for delivering the message to the intended recipient, any dissemination, distribution or copying of the communication is strictly prohibited. If you received the communication in error, please notify the sender immediately by replying to this message and deleting the message and any accompanying files from your system. If, due to the security risks, you do not wish to receive further communications via e-mail, please reply to this message and inform the sender that you do not wish to receive further e-mail from the sender. --------------------------------------------------------------------- |
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Craig Brideau |
Re: spectral versus well filtered in plants
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In reply to this post by Boswell, Carl A - (cboswell)
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. |
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Rosemary.White |
Re: spectral versus well filtered in plants
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In reply to this post by Kurt Thorn
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 >> > |
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Craig Brideau |
Re: spectral versus well filtered in plants
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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 |
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O'Toole, P |
Re: spectral versus well filtered in plants
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In reply to this post by Christian-103
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 Tel : +44 (0)1904 328722 Fax : +44 (0)1904 328804 email : [hidden email] www.york.ac.uk/biology/tf |
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