probe for MP excitation
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Koo, Lily (NIH/NIAID) [E] |
probe for MP excitation
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Dear all,
I wonder if anyone had experience in trying dyes that are suitable for MP excitation and emit in the blue and/or far-red spectrum? Would any Alexa or LiveTracker dyes work well there? Thanks, Lily |
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MP excited far-red dyes are not a starter since the blocking filters
will block the signal as well. Blue should be easy - after all everyone uses DAPI and Hoechst. I've also imaged blue FPs - not yet cloned, though. Guy Optical Imaging Techniques in Cell Biology by Guy Cox CRC Press / Taylor & Francis http://www.guycox.com/optical.htm ______________________________________________ Associate Professor Guy Cox, MA, DPhil(Oxon) Electron Microscope Unit, Madsen Building F09, University of Sydney, NSW 2006 Phone +61 2 9351 3176 Fax +61 2 9351 7682 Mobile 0413 281 861 ______________________________________________ http://www.guycox.net -----Original Message----- From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Koo, Lily (NIH/NIAID) [E] Sent: Thursday, 21 January 2010 1:56 PM To: [hidden email] Subject: probe for MP excitation Dear all, I wonder if anyone had experience in trying dyes that are suitable for MP excitation and emit in the blue and/or far-red spectrum? Would any Alexa or LiveTracker dyes work well there? Thanks, Lily No virus found in this incoming message. Checked by AVG - www.avg.com Version: 9.0.730 / Virus Database: 270.14.151/2633 - Release Date: 01/21/10 06:18:00 |
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Good point!
I've been reading up on a few promising reagents recently only to realise that the blocking filter rules them out. But it does depends a little what you define as "far-red"... :) Invitrogen have several products that they call "far-red" that have plenty of emission <700nm (where I'm guessing most MP blocking filters sit?) I've read at least one paper that uses AlexaFluor 660 but that looks to be optimal with OPO excitation at 1100nm (Andresen et al, Current Opinion in Biotechnology 2009, 20:1-9). I've spoken to a couple of people in recent weeks who've used AlexaFluor 647 with standard Ti-Sa excitation. We've only just started trying it here and had mixed success (not entirely clear why yet). You do have to look closely at the full transmission profiles of all the filters (especially dichroics if you are using them for mutli-channel imaging) in the pathway. Regards, Adrian Smith Centenary Institute, Sydney, Australia On 21/01/2010, at 10:51 PM, Guy Cox wrote: > MP excited far-red dyes are not a starter since the blocking filters > will block the signal as well. Blue should be easy - after all everyone > uses DAPI and Hoechst. I've also imaged blue FPs - not yet cloned, > though. > > Guy > > Optical Imaging Techniques in Cell Biology > by Guy Cox CRC Press / Taylor & Francis > http://www.guycox.com/optical.htm > ______________________________________________ > Associate Professor Guy Cox, MA, DPhil(Oxon) > Electron Microscope Unit, Madsen Building F09, > University of Sydney, NSW 2006 > > Phone +61 2 9351 3176 Fax +61 2 9351 7682 > Mobile 0413 281 861 > ______________________________________________ > http://www.guycox.net > > > > -----Original Message----- > From: Confocal Microscopy List [mailto:[hidden email]] > On Behalf Of Koo, Lily (NIH/NIAID) [E] > Sent: Thursday, 21 January 2010 1:56 PM > To: [hidden email] > Subject: probe for MP excitation > > Dear all, > > I wonder if anyone had experience in trying dyes that are suitable for > MP excitation and emit in the blue and/or far-red spectrum? Would any > Alexa or LiveTracker dyes work well there? > > Thanks, > > Lily > > No virus found in this incoming message. > Checked by AVG - www.avg.com > Version: 9.0.730 / Virus Database: 270.14.151/2633 - Release Date: > 01/21/10 06:18:00 |
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It depends on what filters are in your system. Since most Ti-S lasers
can be tuned shorter than 700nm you are unlikely to be able to get anything longer than 650nm in the detectors. But of course if you are willing to take on yourself the responsibility to fit custom filters you could go lower - just remember if you tune your laser too short you will fry your PMTs! Guy Optical Imaging Techniques in Cell Biology by Guy Cox CRC Press / Taylor & Francis http://www.guycox.com/optical.htm ______________________________________________ Associate Professor Guy Cox, MA, DPhil(Oxon) Electron Microscope Unit, Madsen Building F09, University of Sydney, NSW 2006 Phone +61 2 9351 3176 Fax +61 2 9351 7682 Mobile 0413 281 861 ______________________________________________ http://www.guycox.net -----Original Message----- From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Adrian Smith Sent: Thursday, 21 January 2010 11:13 PM To: [hidden email] Subject: Re: probe for MP excitation Good point! I've been reading up on a few promising reagents recently only to realise that the blocking filter rules them out. But it does depends a little what you define as "far-red"... :) Invitrogen have several products that they call "far-red" that have plenty of emission <700nm (where I'm guessing most MP blocking filters sit?) I've read at least one paper that uses AlexaFluor 660 but that looks to be optimal with OPO excitation at 1100nm (Andresen et al, Current Opinion in Biotechnology 2009, 20:1-9). I've spoken to a couple of people in recent weeks who've used AlexaFluor 647 with standard Ti-Sa excitation. We've only just started trying it here and had mixed success (not entirely clear why yet). You do have to look closely at the full transmission profiles of all the filters (especially dichroics if you are using them for mutli-channel imaging) in the pathway. Regards, Adrian Smith Centenary Institute, Sydney, Australia On 21/01/2010, at 10:51 PM, Guy Cox wrote: > MP excited far-red dyes are not a starter since the blocking filters > will block the signal as well. Blue should be easy - after all everyone > uses DAPI and Hoechst. I've also imaged blue FPs - not yet cloned, > though. > > Guy > > Optical Imaging Techniques in Cell Biology > by Guy Cox CRC Press / Taylor & Francis > http://www.guycox.com/optical.htm > ______________________________________________ > Associate Professor Guy Cox, MA, DPhil(Oxon) > Electron Microscope Unit, Madsen Building F09, > University of Sydney, NSW 2006 > > Phone +61 2 9351 3176 Fax +61 2 9351 7682 > Mobile 0413 281 861 > ______________________________________________ > http://www.guycox.net > > > > -----Original Message----- > From: Confocal Microscopy List > On Behalf Of Koo, Lily (NIH/NIAID) [E] > Sent: Thursday, 21 January 2010 1:56 PM > To: [hidden email] > Subject: probe for MP excitation > > Dear all, > > I wonder if anyone had experience in trying dyes that are suitable for > MP excitation and emit in the blue and/or far-red spectrum? Would any > Alexa or LiveTracker dyes work well there? > > Thanks, > > Lily > > No virus found in this incoming message. > Checked by AVG - www.avg.com > Version: 9.0.730 / Virus Database: 270.14.151/2633 - Release Date: > 01/21/10 06:18:00 No virus found in this incoming message. Checked by AVG - www.avg.com Version: 9.0.730 / Virus Database: 270.14.151/2633 - Release Date: 01/21/10 06:18:00 |
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Koo, Lily (NIH/NIAID) [E] |
Re: probe for MP excitation
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Yes, we have in fact two blocking filters - 660nm or 710nm. So something like Alexa 633 or 647 should be detectable in theory. I wonder if any of you have experience exciting them or similar dyes with MP? Some papers seemed to have used A647, but the methods were not clearly described.
What about Alexa 405, Pacific Blue, or other blue dyes? We are trying to maximize the number of dyes that can use in the tissues; more specifically, something in combination with GFP/YFP/CFSE and a red/orange dye such as dsRed/RFP/CMPTX. Any suggestions will be appreciated. Thanks, Lily -----Original Message----- From: Guy Cox [mailto:[hidden email]] Sent: Thursday, January 21, 2010 7:22 AM To: [hidden email] Subject: Re: probe for MP excitation It depends on what filters are in your system. Since most Ti-S lasers can be tuned shorter than 700nm you are unlikely to be able to get anything longer than 650nm in the detectors. But of course if you are willing to take on yourself the responsibility to fit custom filters you could go lower - just remember if you tune your laser too short you will fry your PMTs! Guy Optical Imaging Techniques in Cell Biology by Guy Cox CRC Press / Taylor & Francis http://www.guycox.com/optical.htm ______________________________________________ Associate Professor Guy Cox, MA, DPhil(Oxon) Electron Microscope Unit, Madsen Building F09, University of Sydney, NSW 2006 Phone +61 2 9351 3176 Fax +61 2 9351 7682 Mobile 0413 281 861 ______________________________________________ http://www.guycox.net -----Original Message----- From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Adrian Smith Sent: Thursday, 21 January 2010 11:13 PM To: [hidden email] Subject: Re: probe for MP excitation Good point! I've been reading up on a few promising reagents recently only to realise that the blocking filter rules them out. But it does depends a little what you define as "far-red"... :) Invitrogen have several products that they call "far-red" that have plenty of emission <700nm (where I'm guessing most MP blocking filters sit?) I've read at least one paper that uses AlexaFluor 660 but that looks to be optimal with OPO excitation at 1100nm (Andresen et al, Current Opinion in Biotechnology 2009, 20:1-9). I've spoken to a couple of people in recent weeks who've used AlexaFluor 647 with standard Ti-Sa excitation. We've only just started trying it here and had mixed success (not entirely clear why yet). You do have to look closely at the full transmission profiles of all the filters (especially dichroics if you are using them for mutli-channel imaging) in the pathway. Regards, Adrian Smith Centenary Institute, Sydney, Australia On 21/01/2010, at 10:51 PM, Guy Cox wrote: > MP excited far-red dyes are not a starter since the blocking filters > will block the signal as well. Blue should be easy - after all everyone > uses DAPI and Hoechst. I've also imaged blue FPs - not yet cloned, > though. > > Guy > > Optical Imaging Techniques in Cell Biology > by Guy Cox CRC Press / Taylor & Francis > http://www.guycox.com/optical.htm > ______________________________________________ > Associate Professor Guy Cox, MA, DPhil(Oxon) > Electron Microscope Unit, Madsen Building F09, > University of Sydney, NSW 2006 > > Phone +61 2 9351 3176 Fax +61 2 9351 7682 > Mobile 0413 281 861 > ______________________________________________ > http://www.guycox.net > > > > -----Original Message----- > From: Confocal Microscopy List > On Behalf Of Koo, Lily (NIH/NIAID) [E] > Sent: Thursday, 21 January 2010 1:56 PM > To: [hidden email] > Subject: probe for MP excitation > > Dear all, > > I wonder if anyone had experience in trying dyes that are suitable for > MP excitation and emit in the blue and/or far-red spectrum? Would any > Alexa or LiveTracker dyes work well there? > > Thanks, > > Lily > > No virus found in this incoming message. > Checked by AVG - www.avg.com > Version: 9.0.730 / Virus Database: 270.14.151/2633 - Release Date: > 01/21/10 06:18:00 No virus found in this incoming message. Checked by AVG - www.avg.com Version: 9.0.730 / Virus Database: 270.14.151/2633 - Release Date: 01/21/10 06:18:00 |
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We had good success visualizing Coumarin with 2P excitation. This was a while ago, and I don't remember the details.
I also remember that far red dyes tended to bleach quite fast with 2P excitation, which I attributed, and this was just a guess, to the fact that the excitation spectrum of these dyes may extend into the wavelengths used by 2P excitation, and therefore they may just be excited (and bleached) through the normal one photon mode. For example, if you are using 750 nm to excite DAPI in 2P mode, you would at the same time be exciting Alexa 680 at about 3% max efficiency (in one photon mode), but with intensities several hundred times greater than you would with a 633 laser, so overall your Alexa dye would be getting excited (and bleached) far more than when you are imaging it in normal (one photon) mode with a conventional 633 laser. Maybe someone could correct me if this doesn't make sense... -- Julio Vazquez Fred Hutchinson Cancer Research Center 1100 Fairview Ave. N., mailstop DE-512 Seattle, WA 98109-1024 == On Jan 21, 2010, at 7:30 AM, Koo, Lily (NIH/NIAID) [E] wrote:
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