It has been my experience that AlexaFluor647 is brighter than AF633
even with a 633 laser. The extinction coefficient of AF647 is more
than 2x that of AF633 so that compensates for the less-than-ideal
laser. In addition our standard "far red" emission filters better
match the emission spectrum of AF647 and I suspect the quantum yield
of AF647 is higher too, though the last time I checked the Invitrogen
website that specification was not given.
-Esteban
2009/11/4 Junya HIROI <[log in to unmask]>:
> Hi Andrea,
>
> I actually did 5-color staining on a Zeiss LSM510, with
> Alexa 405, Alexa 430, Alexa 488, Alexa 555 and Alexa 647.
>
> http://jeb.biologists.org/cgi/content/full/211/16/2584
> (Figs 6 and 8 are 5-color staining)
>
> I used Alexa 405 and 430 in the above paper, but later I found that Pacific
> Blue is better than Alexa 405, and Pacific Orange is better than Alexa 430
> (brighter and more stable, although not as stable as other Alexa dyes, such
> as Alexa 488, 555 and 647). I did not check Marina blue yet.
>
> I did not compare Alexa 647 and Alexa 633, but I have heard that Alexa 647
> is better than Alexa 633, even if with 633 nm laser.
>
> Best wishes,
>
> Junya
>
>
> On 11/4/09, at 7:16 , Andrea Hooper wrote:
>
>> Dear All,
>>
>> I need some advice in setting up for a very important experiment coming
>> up. Has anyone used the fluorophore PerCP CY5.5 with success for confocal
>> microscopy? Any experience with autofluorescence in this wavelength or
>> whether this fluorophore is a severe bleacher etc.?
>>
>> I want to do 5 or 6 color on a Zeiss LSM710 and am thinking about the
>> following, I would love your feedback.
>> ? Alexa405
>> ? Alexa430
>> ? GFP
>> ? RFP
>> ? Alexa633
>> ? PerCP CY5.5
>> Thank you in advance,
>> Andrea
>
> ___________________________________________
> Junya HIROI
> Department of Anatomy
> St. Marianna University School of Medicine
> 2-16-1 Sugao, Miyamae-ku
> Kawasaki 216-8511, JAPAN
> phone: +81-44-977-8111 ext.3625
> fax: +81-44-976-7083
> e-mail: [log in to unmask]
> ___________________________________________
>
--
G. Esteban Fernandez, Ph.D.
Associate Director
Molecular Cytology Core Facility
University of Missouri
120 Bond Life Sciences Center
Columbia, MO 65211
http://www.biotech.missouri.edu/mcc
(573)882-4895
(573)884-9676 fax
|
