Michal Gdula-2 |
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** Dear Confocalists, I am in desperate need for a new fuorophores for my multicolor FISH- immunofluorescence experiments. I am using at the moment DAPI, Alxea488, Cy3, Texas Red and Cy5. I use for imaging Zeiss microscope LSM510meta. I am thinking about purchasing antibodies conjugated with Alexa 680 or Alexa 700. According to olipmpus fluorophore tutorial web page my 633 nm laser should be able to excite both although its quite far away from excitation maximum. Has anybody used successfully any of these fluorophores with LSM510 meta? Is it easy to separate them from Cy5 using meta detector? Molecular mass of of these fluorophores is relatively high (1.2-1.4 kDa) compared with the fluorophores which I am using right now, will it have effect on the antibody penetration ? Kind regards, Michal Gdula PhD student Bradford University Uk |
George McNamara |
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** Hi Michal, You have a META spectral detector - use the spectral unmixing capability. If necessary, you can acquire sequentially with different lasers and combine the images later. PMT's have poor quantum efficiency in the far red, so even if Cy5.5, AF680 or AF700 dye, or QD705 quantum dot conjugates work well at the specimen level the signal might be weak. QD705 might have an advantage in being excitable with the same laser as DAPI. Tsurui et al published 7 color immunofluorescence eleven years ago: Tsurui H, Nishimura H, Hattori S, Hirose S, Okumura K, Shirai T. Seven-color fluorescence imaging of tissue samples based on Fourier spectroscopy and singular value decomposition. </pubmed/10769049> J Histochem Cytochem. 2000 May;48(5):653-62. PMID: 10769049 You might take a dye from them and add AF532 to your mix (AF546 is not as useful a dye as AF555 or AF568). When you have an opportunity, replacing Cy3 with any of Cy3B (B=Brighter), AF555 or AF568 should increase brightness. If any of your reagents are dim, you can use tyramide signal amplification to boost the signal between 10x and 100x. PerkinElmer has a limited TSA line-up, Invitrogen/Molecular probes has many fluorescent TSA kits. Anothre possibility (instead of NIRF or AF532) would be Lucifer Yellow ... AF430, excited with the same laser as DAPI. If you need to see spectra of any of these, see http://www.mcb.arizona.edu/ipc/fret/index.html If you want the spectra data, see the XLSX file inside the PubSpectra zip file at the top of http://sylvester.org/research/shared-resources/laboratory-resources/analytical-imaging-core-facility/analytical-imaging-core-facility-links-forms/pubspectra-data Best wishes, George p.s. there are over 700 spectral karyotyping papers that use five FISH/immuno reagents acquired simultaneously plus DAPI (acquired separately with a DAPI filter set). The dye-detection reagents used include DNA probes conjugated with Rhodamine 110 (green fluorophore), spectrum orange, spectrum Red (Texas Red), biotin and digoxigenin, with detection reagents Cy5-streptavidin and Cy5.5-anti-mouse/mouse anti-dig. I will guess here that over 10,000 specimens have been acquired and analyzed for clinical + research use. The META detector ought to be able to handle five color unmxing as well as the SKY camera. On 2/20/2011 9:01 AM, Michal Gdula wrote: > ***** > To join, leave or search the confocal microscopy listserv, go to: > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy > ***** > > Dear Confocalists, > > I am in desperate need for a new fuorophores for my multicolor FISH- > immunofluorescence experiments. I am using at the moment DAPI, Alxea488, Cy3, > Texas Red and Cy5. I use for imaging Zeiss microscope LSM510meta. > I am thinking about purchasing antibodies conjugated with Alexa 680 or Alexa > 700. According to olipmpus fluorophore tutorial web page my 633 nm laser should > be able to excite both although its quite far away from excitation maximum. > Has anybody used successfully any of these fluorophores with LSM510 meta? > Is it easy to separate them from Cy5 using meta detector? > Molecular mass of of these fluorophores is relatively high (1.2-1.4 kDa) compared > with the fluorophores which I am using right now, will it have effect on the > antibody penetration ? > > Kind regards, > > Michal Gdula > PhD student > Bradford University > Uk > > -- George McNamara, PhD Analytical Imaging Core Facility University of Miami |
Julio Vazquez |
In reply to this post by Michal Gdula-2
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** Michal, I have not tried those dyes. However, the range of the META detector on our system goes up to ~ 720 nm (according to the graphical display in the LMETA control panel in LSM software). According to this, Alexa 680 (or Cy5.5) should be detected about twice as efficiently as Alexa 700. Both Alexa 680, Cy5.5, and 700 should be distinct enough from Cy5 for the META to separate them, again in theory, so my best bet would be 680 or Cy5.5, with 680 being possibly the brighter and more stable choice. Also, as an alternative, you may consider using dyes with large stokes shift, i.e. separate your dyes based on excitation rather than emission, but I have no such dye in particular to recommend. -- Julio Vazquez Fred Hutchinson Cancer Research Center Seattle, WA 98109-1024 http://www.fhcrc.org == On Feb 20, 2011, at 6:01 AM, Michal Gdula wrote: > ***** > To join, leave or search the confocal microscopy listserv, go to: > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy > ***** > > Dear Confocalists, > > I am in desperate need for a new fuorophores for my multicolor FISH- > immunofluorescence experiments. I am using at the moment DAPI, > Alxea488, Cy3, > Texas Red and Cy5. I use for imaging Zeiss microscope LSM510meta. > I am thinking about purchasing antibodies conjugated with Alexa 680 > or Alexa > 700. According to olipmpus fluorophore tutorial web page my 633 nm > laser should > be able to excite both although its quite far away from excitation > maximum. > Has anybody used successfully any of these fluorophores with LSM510 > meta? > Is it easy to separate them from Cy5 using meta detector? > Molecular mass of of these fluorophores is relatively high (1.2-1.4 > kDa) compared > with the fluorophores which I am using right now, will it have > effect on the > antibody penetration ? > > Kind regards, > > Michal Gdula > PhD student > Bradford University > Uk |
Michal Gdula-2 |
In reply to this post by Michal Gdula-2
*****
To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** Thank you Julio for the answer, I will probably use Alexa 680 as a best compromise between distance from optimum of excitation ( my laser has 633 nm) and the distance from Cy5. I am searching also on the other end of the spectrum - I can give up DAPI and use Alexa 350, however my experience so far is that it gives weak signals. I am restricted again with argon UV laser 360 nm (so unfortunatelly I can not use DEAC)- any advice fro strong dye which would fit, or on succesful use of ALexa 350 for imaging of FISH signals with confocal microscope would be appreciated. Michal |
Michal Gdula-2 |
In reply to this post by Michal Gdula-2
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** Thank you George for such a comprehensive answer, I have already tried spectral unmixing already and I got dissapointed. Correct me if I am wrong but it works nicely only if you have objects stained with overlapping dyes labelled with similar strength. I tried to unmix Alexa 514 from Alexa 488 and Cy3. Alexa 514 was labelling nucleoli and the staining was strong and visible in red and green channel. And two other dyes labelled other structure which did not give such a strong signal mostly because of their structure and quantity. I was not able to resolve these dyes. I am aware of the literarture on multicolor FISH with epifluorescent microscope, however as far as I know many of dyes used there don't work well with confocal microscope. I am also considering the fluorophores used in the paper of Tsurui et al, however its worth to mention that he used epifluorescent microscope for imaging combined with spectral imager. I am limited to my LSM510 meta. The Alexa 430 looks interesting - its emission seem to overlap with AF488 but should not be excited almost at all with 488 nm laser ( at least according to olimpus website (http://www.olympusfluoview.com/java/dualprobes/index.html). I am searching also for some blue fluorophore which would be bright, excited efficiently with 364 nm laser and possibility to conjugate with dUTP. Thank you one more time for your advice, Michal |
Jacqueline Ross |
In reply to this post by Michal Gdula-2
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** Hi Michal, Alexa 350 is quite a weak fluorophore, however we do have people here using it successfully when used in high concentration (e.g. 1/50). They are using it for standard immunohistochemistry rather than FISH. You do need to take extra care with regard to photobleaching however. For example, don't spend a lot of time viewing it by eye down the microscope prior to confocal imaging. I think Mike Ignatius/Invitrogen has previously advised that Pacific Blue is a better choice than Alexa 350 and if you check out the archives you will probably find his email regarding the hierarchy of their UV excitable fluorophores. Kind regards, Jacqui Jacqueline Ross Biomedical Imaging Microscopist Biomedical Imaging Research Unit School of Medical Sciences Faculty of Medical & Health Sciences The University of Auckland Private Bag 92019 Auckland, NEW ZEALAND Tel: 64 9 373 7599 Ext 87438 Fax: 64 9 373 7484 http://www.fmhs.auckland.ac.nz/sms/biru/ -----Original Message----- From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Michal Gdula Sent: Monday, 21 February 2011 8:04 a.m. To: [hidden email] Subject: Re: Alexa 680 and 700 and Zeiss LSM510meta ***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** Thank you Julio for the answer, I will probably use Alexa 680 as a best compromise between distance from optimum of excitation ( my laser has 633 nm) and the distance from Cy5. I am searching also on the other end of the spectrum - I can give up DAPI and use Alexa 350, however my experience so far is that it gives weak signals. I am restricted again with argon UV laser 360 nm (so unfortunatelly I can not use DEAC)- any advice fro strong dye which would fit, or on succesful use of ALexa 350 for imaging of FISH signals with confocal microscope would be appreciated. Michal |
Ignatius, Mike-2 |
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** Hi Michael, We have two other dyes that would work with that 360nm argon laser line, Marina Blue and Alexa Fluor 405. Pac Blue recommended by Jacqui below, is not much for 360 ex (19%), preferring 405 ex much more. And the archived statement on UV dye selection referred to won't have info comparing any of these to Alexa Fluor 350. We didn't compare AF 350 to these others - sorry. While its name, AF405, implies otherwise, this dye absorbs both 360nm and 405nm lines quite well. Nearly 60% for the 360 line. Try our spectraviewer to see the data for Alexa Fluor 405, as Olympus doesn't have this dye in their data base currently. If you are viewing in PBS, AF 405 is the better dye, while in Prolong Gold mounted samples, Marina is a bit better. However your 360 laser, scan speeds, magnification, mountant choice and other imaging settings may well alter those predictions. All UV ex dyes fade very fast - except Qdots of course. But no Qdot equivalent for this emission range. Obviously and especially for UV dyes, turn up detector gain, turn down the excitation power, dark adapt the eyes and focus /compose fast. Best of luck, Mike Ignatius with help and data from Jason Kilgore Molecular Probes, Life Technologies PS. I changed the subject line for future archivists. -----Original Message----- From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Jacqui Ross Sent: Sunday, February 20, 2011 2:44 PM To: [hidden email] Subject: Re: Alexa 680 and 700 and Zeiss LSM510meta ***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** Hi Michal, Alexa 350 is quite a weak fluorophore, however we do have people here using it successfully when used in high concentration (e.g. 1/50). They are using it for standard immunohistochemistry rather than FISH. You do need to take extra care with regard to photobleaching however. For example, don't spend a lot of time viewing it by eye down the microscope prior to confocal imaging. I think Mike Ignatius/Invitrogen has previously advised that Pacific Blue is a better choice than Alexa 350 and if you check out the archives you will probably find his email regarding the hierarchy of their UV excitable fluorophores. Kind regards, Jacqui Jacqueline Ross Biomedical Imaging Microscopist Biomedical Imaging Research Unit School of Medical Sciences Faculty of Medical & Health Sciences The University of Auckland Private Bag 92019 Auckland, NEW ZEALAND Tel: 64 9 373 7599 Ext 87438 Fax: 64 9 373 7484 http://www.fmhs.auckland.ac.nz/sms/biru/ -----Original Message----- From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Michal Gdula Sent: Monday, 21 February 2011 8:04 a.m. To: [hidden email] Subject: Re: Alexa 680 and 700 and Zeiss LSM510meta ***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** Thank you Julio for the answer, I will probably use Alexa 680 as a best compromise between distance from optimum of excitation ( my laser has 633 nm) and the distance from Cy5. I am searching also on the other end of the spectrum - I can give up DAPI and use Alexa 350, however my experience so far is that it gives weak signals. I am restricted again with argon UV laser 360 nm (so unfortunatelly I can not use DEAC)- any advice fro strong dye which would fit, or on succesful use of ALexa 350 for imaging of FISH signals with confocal microscope would be appreciated. Michal |
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