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
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