Hi Olivier,

We have multiple-labelled with Cy5 and Cy5.5, the spectral confocals
can separate them quite well after a bit of fiddling around.  Our
(now fairly old) Leica SP2 does this just fine.

cheers,
Rosemary

Rosemary White
CSIRO Plant Industry
GPO Box 1600
Canberra, ACT 2601
Australia

ph 61 2 6246 5475
fx 61 2 6246 5334
________________________________________
From: Confocal Microscopy List [[log in to unmask]] On Behalf Of Alison North [[log in to unmask]]
Sent: Thursday, 6 November 2008 5:02 a.m.
To: [log in to unmask]
Subject: Re: Alexa 750

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