One word of caution regarding the Qdots is that since they are excited by all lasers you canīt
do sequential scanning (or at least you wonīt 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 Iīve 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: [log in to unmask]
fax: +46-8-335724
--------------------------------------
----- Original Message -----
From: Guillermo Palchik <[log in to unmask]>
Date: Wednesday, November 5, 2008 8:11 pm
Subject: Re: Alexa 750
To: [log in to unmask]
> 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
> [log in to unmask]
>
|
