http://confocal-microscopy-list.275.s1.nabble.com/Alexa-750-tp1459204p1461256.html
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.