http://confocal-microscopy-list.275.s1.nabble.com/Alexa-750-tp1459204p1461586.html
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.
> 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]
>