Alexa 750
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Hello
We are using DAPI, FITC, Cy3 and Cy5 for immunolabelling. We want to use a fifth label and have found that Alexa750 seems to be a good possibility. We are working on Zeiss LSM 510 Meta. Does anyone have been using this dye? Is it really possible to discriminate Cy3 from Alexa750? I will appreciate any advices. Thanks |
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Hi Olivier, I doubt the META detector can read emissions past 720-750 nm. Also, if you look at the excitation/emission spectra of Alexa 750, such as here: you will see that with standard confocal lasers (488, 543/561/633), you will achieve an excitation efficiency of at most 10%. Finally, if you want to use a conventional detector, you would need an appropriate emission filter on your confocal, such as a longpass 730 or higher, which is probably not standard, and you probably would have rather poor detection efficiency, unless you have special IR PMTs... One suggestion would be to use a dye in the red range that you can spectrally separate from Cy3, or maybe use something with a large stokes shift, such as a Qdot 605 or similar, that you can excite at around 450 or 488, and detect in the red channel, and therefore can be separated from Cy3 based on the different excitation efficiency. Something like Cy5-PE might work too (Excitation at 488, emission at 680) -- Julio Vazquez Fred Hutchinson Cancer Research Center Seattle, WA 98109-1024 On Nov 5, 2008, at 12:32 AM, Olivier Bardot wrote:
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Hi Olivier,
I agree with Julio that the Alexa 750 may not be the best choice. One possibility is to add in another red dye that could be separated from the Cy3 using the META detector - either Alexa 568 or Rhodamine Red-X should work fine, in our experience. Another is to add a second green colour and separate that from the FITC using the META - or maybe swap from FITC to AF 488 if possible? - we have separated AF 488 and AF 514 nicely using the META, and the META detector is anyway most sensitive in the green emission region. You would need to tweak the labelling conditions so that the colours you are using with the META detector are pretty well balanced, and we also tend to prefer collecting the regular channels in one scan and the fluorochromes to be unmixed by the META in a separate scan.- e.g. if you were using two reds, set up a DAPI/FITC/Cy5 regular scan, followed by a lambda stack just imaging the reds. Then combine the results after unmixing. If you try to collect everything on one lambda scan, you will have the problem of having to uncheck the window before and after each of the additional laser lines in the lambda stack when you do the unmixing. I hope this makes sense, if not please feel free to contact me offline. Best, Alison Julio Vazquez wrote: > = > Hi Olivier, > > I doubt the META detector can read emissions past 720-750 nm. Also, if > you look at the excitation/emission spectra of Alexa 750, such as here: > > http://www.bdbiosciences.com/spectra/ > > you will see that with standard confocal lasers (488, 543/561/633), > you will achieve an excitation efficiency of at most 10%. Finally, if > you want to use a conventional detector, you would need an appropriate > emission filter on your confocal, such as a longpass 730 or higher, > which is probably not standard, and you probably would have rather > poor detection efficiency, unless you have special IR PMTs... > > One suggestion would be to use a dye in the red range that you can > spectrally separate from Cy3, or maybe use something with a large > stokes shift, such as a Qdot 605 or similar, that you can excite at > around 450 or 488, and detect in the red channel, and therefore can be > separated from Cy3 based on the different excitation efficiency. > Something like Cy5-PE might work too (Excitation at 488, emission at 680) > > > -- > Julio Vazquez > Fred Hutchinson Cancer Research Center > Seattle, WA 98109-1024 > > > http://www.fhcrc.org/ > > > > On Nov 5, 2008, at 12:32 AM, Olivier Bardot wrote: > >> Hello >> We are using DAPI, FITC, Cy3 and Cy5 for immunolabelling. We want to >> use a >> fifth label and have found that Alexa750 seems to be a good possibility. >> We are working on Zeiss LSM 510 Meta. >> Does anyone have been using this dye? >> Is it really possible to discriminate Cy3 from Alexa750? >> I will appreciate any advices. >> Thanks > -- Alison J. North, Ph.D., Research Assistant Professor and Director of the Bio-Imaging Resource Center, The Rockefeller University, 1230 York Avenue, New York, NY 10065. Tel: office ++ 212 327 7488 Tel: lab ++ 212 327 7486 Fax: ++ 212 327 7489 |
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One could also try a long-Stokes-shift fluor such as Chromeo 494 that
is excited together with AF 488 or FITC, but that emits in the mid 600s. No unmixing required when doing sequential scans. >Hi Olivier, > >I agree with Julio that the Alexa 750 may not be the best choice. >One possibility is to add in another red dye that could be separated >from the Cy3 using the META detector - either Alexa 568 or Rhodamine >Red-X should work fine, in our experience. Another is to add a >second green colour and separate that from the FITC using the META - >or maybe swap from FITC to AF 488 if possible? - we have separated >AF 488 and AF 514 nicely using the META, and the META detector is >anyway most sensitive in the green emission region. You would need >to tweak the labelling conditions so that the colours you are using >with the META detector are pretty well balanced, and we also tend to >prefer collecting the regular channels in one scan and the >fluorochromes to be unmixed by the META in a separate scan.- e.g. if >you were using two reds, set up a DAPI/FITC/Cy5 regular scan, >followed by a lambda stack just imaging the reds. Then combine the >results after unmixing. If you try to collect everything on one >lambda scan, you will have the problem of having to uncheck the >window before and after each of the additional laser lines in the >lambda stack when you do the unmixing. > >I hope this makes sense, if not please feel free to contact me offline. > >Best, >Alison > >Julio Vazquez wrote: >>= >>Hi Olivier, >>I doubt the META detector can read emissions past 720-750 nm. Also, >>if you look at the excitation/emission spectra of Alexa 750, such >>as here: >> >>http://www.bdbiosciences.com/spectra/ >> >>you will see that with standard confocal lasers (488, 543/561/633), >>you will achieve an excitation efficiency of at most 10%. Finally, >>if you want to use a conventional detector, you would need an >>appropriate emission filter on your confocal, such as a longpass >>730 or higher, which is probably not standard, and you probably >>would have rather poor detection efficiency, unless you have >>special IR PMTs... >> >>One suggestion would be to use a dye in the red range that you can >>spectrally separate from Cy3, or maybe use something with a large >>stokes shift, such as a Qdot 605 or similar, that you can excite at >>around 450 or 488, and detect in the red channel, and therefore can >>be separated from Cy3 based on the different excitation efficiency. >>Something like Cy5-PE might work too (Excitation at 488, emission >>at 680) >> >> >>-- >>Julio Vazquez >>Fred Hutchinson Cancer Research Center >>Seattle, WA 98109-1024 >> >> >>http://www.fhcrc.org/ >> >> >> >>On Nov 5, 2008, at 12:32 AM, Olivier Bardot wrote: >> >>>Hello >>>We are using DAPI, FITC, Cy3 and Cy5 for immunolabelling. We want to use a >>>fifth label and have found that Alexa750 seems to be a good possibility. >>>We are working on Zeiss LSM 510 Meta. >>>Does anyone have been using this dye? >>>Is it really possible to discriminate Cy3 from Alexa750? >>>I will appreciate any advices. >>>Thanks >> > >-- >Alison J. North, Ph.D., >Research Assistant Professor and Director of the Bio-Imaging Resource Center, >The Rockefeller University, >1230 York Avenue, >New York, >NY 10065. >Tel: office ++ 212 327 7488 >Tel: lab ++ 212 327 7486 >Fax: ++ 212 327 7489 -- Robert J. Palmer Jr., Ph.D. Natl Inst Dental Craniofacial Res - Natl Insts Health Oral Infection and Immunity Branch Bldg 30, Room 310 30 Convent Drive Bethesda MD 20892 ph 301-594-0025 fax 301-402-0396 |
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Another possibility, if you already are thinking of having 5 colors,
you should start considering using quantum dots... On Nov 5, 2008, at 1:20 PM, Robert J. Palmer Jr. wrote: > One could also try a long-Stokes-shift fluor such as Chromeo 494 > that is excited together with AF 488 or FITC, but that emits in the > mid 600s. No unmixing required when doing sequential scans. > >> Hi Olivier, >> >> I agree with Julio that the Alexa 750 may not be the best choice. >> One possibility is to add in another red dye that could be >> separated from the Cy3 using the META detector - either Alexa 568 >> or Rhodamine Red-X should work fine, in our experience. Another is >> to add a second green colour and separate that from the FITC using >> the META - or maybe swap from FITC to AF 488 if possible? - we have >> separated AF 488 and AF 514 nicely using the META, and the META >> detector is anyway most sensitive in the green emission region. >> You would need to tweak the labelling conditions so that the >> colours you are using with the META detector are pretty well >> balanced, and we also tend to prefer collecting the regular >> channels in one scan and the fluorochromes to be unmixed by the >> META in a separate scan.- e.g. if you were using two reds, set up a >> DAPI/FITC/Cy5 regular scan, followed by a lambda stack just imaging >> the reds. Then combine the results after unmixing. If you try to >> collect everything on one lambda scan, you will have the problem of >> having to uncheck the window before and after each of the >> additional laser lines in the lambda stack when you do the unmixing. >> >> I hope this makes sense, if not please feel free to contact me >> offline. >> >> Best, >> Alison >> >> Julio Vazquez wrote: >>> = >>> Hi Olivier, >>> I doubt the META detector can read emissions past 720-750 nm. >>> Also, if you look at the excitation/emission spectra of Alexa 750, >>> such as here: >>> >>> http://www.bdbiosciences.com/spectra/ >>> >>> you will see that with standard confocal lasers (488, >>> 543/561/633), you will achieve an excitation efficiency of at most >>> 10%. Finally, if you want to use a conventional detector, you >>> would need an appropriate emission filter on your confocal, such >>> as a longpass 730 or higher, which is probably not standard, and >>> you probably would have rather poor detection efficiency, unless >>> you have special IR PMTs... >>> >>> One suggestion would be to use a dye in the red range that you can >>> spectrally separate from Cy3, or maybe use something with a large >>> stokes shift, such as a Qdot 605 or similar, that you can excite >>> at around 450 or 488, and detect in the red channel, and therefore >>> can be separated from Cy3 based on the different excitation >>> efficiency. Something like Cy5-PE might work too (Excitation at >>> 488, emission at 680) >>> >>> >>> -- >>> Julio Vazquez >>> Fred Hutchinson Cancer Research Center >>> Seattle, WA 98109-1024 >>> >>> >>> http://www.fhcrc.org/ >>> >>> >>> >>> On Nov 5, 2008, at 12:32 AM, Olivier Bardot wrote: >>> >>>> Hello >>>> We are using DAPI, FITC, Cy3 and Cy5 for immunolabelling. We want >>>> to use a >>>> fifth label and have found that Alexa750 seems to be a good >>>> possibility. >>>> We are working on Zeiss LSM 510 Meta. >>>> Does anyone have been using this dye? >>>> Is it really possible to discriminate Cy3 from Alexa750? >>>> I will appreciate any advices. >>>> Thanks >>> >> >> -- >> Alison J. North, Ph.D., >> Research Assistant Professor and Director of the Bio-Imaging >> Resource Center, >> The Rockefeller University, >> 1230 York Avenue, >> New York, >> NY 10065. >> Tel: office ++ 212 327 7488 >> Tel: lab ++ 212 327 7486 >> Fax: ++ 212 327 7489 > > > -- > Robert J. Palmer Jr., Ph.D. > Natl Inst Dental Craniofacial Res - Natl Insts Health > Oral Infection and Immunity Branch > Bldg 30, Room 310 > 30 Convent Drive > Bethesda MD 20892 > ph 301-594-0025 > fax 301-402-0396 Guillermo Palchik [hidden email] |
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One word of caution regarding the Qdots is that since they are excited by all lasers you can´t
do sequential scanning (or at least you won´t benefit from it) and thus need to really trust your filter settings. I have however used Qdot605 successfully in combination with alexa488 and alexa647. Nowadays I prefer to use alexa555 instead of the Qdot605, to be able to record individual channels and minimize crosstalk. For four color staining I´ve included a Pacific Blue conjugated antibody, recorded in the dapi channel, to the above combination with ok result. Good luck! /Fredrik -------------------------------------- Fredrik Wermeling, PhD-student Karolinska Institutet Department of Medicine Unit of Clinical Allergy Research L2:04 Karolinska Hospital SE-171 76 Stockholm Sweden phone: +46-8-51776696 mail: [hidden email] fax: +46-8-335724 -------------------------------------- ----- Original Message ----- From: Guillermo Palchik <[hidden email]> Date: Wednesday, November 5, 2008 8:11 pm Subject: Re: Alexa 750 To: [hidden email] > Another possibility, if you already are thinking of having 5 > colors, > you should start considering using quantum dots... > > On Nov 5, 2008, at 1:20 PM, Robert J. Palmer Jr. wrote: > > > One could also try a long-Stokes-shift fluor such as Chromeo 494 > > that is excited together with AF 488 or FITC, but that emits in > the > > mid 600s. No unmixing required when doing sequential scans. > > > >> Hi Olivier, > >> > >> I agree with Julio that the Alexa 750 may not be the best > choice. > >> One possibility is to add in another red dye that could be > >> separated from the Cy3 using the META detector - either Alexa > 568 > >> or Rhodamine Red-X should work fine, in our experience. Another > is > >> to add a second green colour and separate that from the FITC > using > >> the META - or maybe swap from FITC to AF 488 if possible? - we > have > >> separated AF 488 and AF 514 nicely using the META, and the META > >> detector is anyway most sensitive in the green emission region. > > >> You would need to tweak the labelling conditions so that the > >> colours you are using with the META detector are pretty well > >> balanced, and we also tend to prefer collecting the regular > >> channels in one scan and the fluorochromes to be unmixed by the > >> META in a separate scan.- e.g. if you were using two reds, set > up a > >> DAPI/FITC/Cy5 regular scan, followed by a lambda stack just > imaging > >> the reds. Then combine the results after unmixing. If you try > to > >> collect everything on one lambda scan, you will have the problem > of > >> having to uncheck the window before and after each of the > >> additional laser lines in the lambda stack when you do the > unmixing.>> > >> I hope this makes sense, if not please feel free to contact me > >> offline. > >> > >> Best, > >> Alison > >> > >> Julio Vazquez wrote: > >>> = > >>> Hi Olivier, > >>> I doubt the META detector can read emissions past 720-750 nm. > >>> Also, if you look at the excitation/emission spectra of Alexa > 750, > >>> such as here: > >>> > >>> http://www.bdbiosciences.com/spectra/ > >>> > >>> you will see that with standard confocal lasers (488, > >>> 543/561/633), you will achieve an excitation efficiency of at > most > >>> 10%. Finally, if you want to use a conventional detector, you > >>> would need an appropriate emission filter on your confocal, > such > >>> as a longpass 730 or higher, which is probably not standard, > and > >>> you probably would have rather poor detection efficiency, > unless > >>> you have special IR PMTs... > >>> > >>> One suggestion would be to use a dye in the red range that you > can > >>> spectrally separate from Cy3, or maybe use something with a > large > >>> stokes shift, such as a Qdot 605 or similar, that you can > excite > >>> at around 450 or 488, and detect in the red channel, and > therefore > >>> can be separated from Cy3 based on the different excitation > >>> efficiency. Something like Cy5-PE might work too (Excitation at > > >>> 488, emission at 680) > >>> > >>> > >>> -- > >>> Julio Vazquez > >>> Fred Hutchinson Cancer Research Center > >>> Seattle, WA 98109-1024 > >>> > >>> > >>> http://www.fhcrc.org/ > >>> > >>> > >>> > >>> On Nov 5, 2008, at 12:32 AM, Olivier Bardot wrote: > >>> > >>>> Hello > >>>> We are using DAPI, FITC, Cy3 and Cy5 for immunolabelling. We > want > >>>> to use a > >>>> fifth label and have found that Alexa750 seems to be a good > >>>> possibility. > >>>> We are working on Zeiss LSM 510 Meta. > >>>> Does anyone have been using this dye? > >>>> Is it really possible to discriminate Cy3 from Alexa750? > >>>> I will appreciate any advices. > >>>> Thanks > >>> > >> > >> -- > >> Alison J. North, Ph.D., > >> Research Assistant Professor and Director of the Bio-Imaging > >> Resource Center, > >> The Rockefeller University, > >> 1230 York Avenue, > >> New York, > >> NY 10065. > >> Tel: office ++ 212 327 7488 > >> Tel: lab ++ 212 327 7486 > >> Fax: ++ 212 327 7489 > > > > > > -- > > Robert J. Palmer Jr., Ph.D. > > Natl Inst Dental Craniofacial Res - Natl Insts Health > > Oral Infection and Immunity Branch > > Bldg 30, Room 310 > > 30 Convent Drive > > Bethesda MD 20892 > > ph 301-594-0025 > > fax 301-402-0396 > > Guillermo Palchik > [hidden email] > |
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In reply to this post by Olivier Bardot
Re the five-color stuff, one could also try a long-Stokes-shift fluor
such as Chromeo 494 that is excited together with AF 488 or FITC, but that emits in the mid 600s. No unmixing required but presumes your sample can survive sequential scans. -- Robert J. Palmer Jr., Ph.D. Natl Inst Dental Craniofacial Res - Natl Insts Health Oral Infection and Immunity Branch Bldg 30, Room 310 30 Convent Drive Bethesda MD 20892 ph 301-594-0025 fax 301-402-0396 |
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In reply to this post by Olivier Bardot
AF750 has been tried in my lab on a 510 META with no success (633 excitation). As has been mentioned by others, the standard laser lines and emission filters are not suitable. We ended up going on a widefield 'scope with an AF750 filter from Chroma and a metal halide lamp. Even though the output of metal halide and the QE of the camera are both poor at ≥750 nm, we got good images with long integration times (> 1 sec.) on a cooled CCD camera.
-Esteban
On Wed, Nov 5, 2008 at 2:32 AM, Olivier Bardot <[hidden email]> wrote: Hello -- G. Esteban Fernandez, Ph.D. Associate Director Molecular Cytology Core Facility University of Missouri 120 Bond Life Sciences Center Columbia, MO 65211 http://www.biotech.missouri.edu/mcc/ 573-882-4895 573-884-9395 fax |
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Another option is using combinatorial labeling. I don't know if that is
possible for your targets but it will avoid an increase in scan time and you can use the labels you have used before and are familiar with. Mariette ____________________________________________ Dr. M.P.C. Kemner-van de Corput, ____________________________________________ MGC - Dept. of Cell Biology & Genetics Erasmus Medical Center Dr. Molewaterplein 50, 3015 GE Rotterdam POB 2040, 3000 CA Rotterdam, The Netherlands Office: H-Ee751; tel: +31 10 704.3949 Lab: H-Ee710; tel lab: +31 10 704.3315 tel secr: +31 10 704.3169 ____________________________________________ http://www2.eur.nl/fgg/ch1/cellbiology/ http://www.thesis.kemner.biz/ ____________________________________________ Op Wo, 5 november, 2008 11:58 pm, schreef G. Esteban Fernandez: > AF750 has been tried in my lab on a 510 META with no success (633 > excitation). As has been mentioned by others, the standard laser lines > and > emission filters are not suitable. We ended up going on a widefield > 'scope > with an AF750 filter from Chroma and a metal halide lamp. Even though the > output of metal halide and the QE of the camera are both poor at 750 nm, > we > got good images with long integration times (> 1 sec.) on a cooled CCD > camera. > > -Esteban > > On Wed, Nov 5, 2008 at 2:32 AM, Olivier Bardot <[hidden email]> wrote: > >> Hello >> We are using DAPI, FITC, Cy3 and Cy5 for immunolabelling. We want to use >> a >> fifth label and have found that Alexa750 seems to be a good possibility. >> We are working on Zeiss LSM 510 Meta. >> Does anyone have been using this dye? >> Is it really possible to discriminate Cy3 from Alexa750? >> I will appreciate any advices. >> Thanks >> > > > > -- > G. Esteban Fernandez, Ph.D. > Associate Director > Molecular Cytology Core Facility > University of Missouri > 120 Bond Life Sciences Center > Columbia, MO 65211 > > http://www.biotech.missouri.edu/mcc/ > > 573-882-4895 > 573-884-9395 fax > |
 
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Kilgore, Jason |
Re: Alexa 750 - Vendor Response
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In reply to this post by G. Esteban Fernandez
**Vendor Response**
Hi, all,
I should note that we have a 510 META here at Molecular
Probes. Though I don't think I have tried using Alexa Fluor 750 on it,
Qdot 800 is not able to be visualized on that system. Qdot 705, or
anything lower, is able to be imaged, though.
Of course, widefield microscopes work fine with Alexa Fluor
750 or Qdot 800 (with correct filter sets and detectors).
Cheers,
Jason
Jason A. Kilgore
Invitrogen Corporation
Molecular Probes Labeling and Detection
Technologies
Technical Service Scientist
Eugene, OR 97402-9132
phone: (541) 335-0353
fax: (541) 335-0238
Please visit our website: www.probes.invitrogen.com
P Save trees. Please don't print
this e-mail unless you really need to.
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of G. Esteban Fernandez Sent: Wednesday, November 05, 2008 2:58 PM To: [hidden email] Subject: Re: Alexa 750 AF750 has been tried in my lab on a 510 META with no success (633
excitation). As has been mentioned by others, the standard laser lines and
emission filters are not suitable. We ended up going on a widefield 'scope
with an AF750 filter from Chroma and a metal halide lamp. Even though the
output of metal halide and the QE of the camera are both poor at ≥750 nm,
we got good images with long integration times (> 1 sec.) on a cooled CCD
camera.
-Esteban
On Wed, Nov 5, 2008 at 2:32 AM, Olivier Bardot <[hidden email]>
wrote: Hello -- G. Esteban Fernandez, Ph.D. Associate Director Molecular Cytology Core Facility University of Missouri 120 Bond Life Sciences Center Columbia, MO 65211 http://www.biotech.missouri.edu/mcc/ 573-882-4895 573-884-9395 fax |
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