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Scott, 

Exciting near the peak is good, but detection efficiency and properties of the dye are also important. Under Alexa dyes in Wikipedia (or the Molecular Probes web site), the quantum yield of Alexa 555 is listed as 0.1, while Alexa 568 is listed as 0.69, so even considering the lower excitation efficiency (about 50%) and the somewhat lower extinction coefficient, Alexa 568 should still be about 2-2.5 times brighter than A555. Or put another way, you could get the same signal with about half the laser intensity (and therefore less photobleaching).

http://en.wikipedia.org/wiki/Alexa_Fluor 

JulioVazquez
Fred Hutchinson Cancer Research Center
Seattle, WA

----- Original Message -----
From: "Scott Wong" <[log in to unmask]>
To: [log in to unmask]
Sent: Thursday, July 4, 2013 12:57:32 PM
Subject: Re: Alexa Fluor 532 with 543nm laser

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Hi Craig,
Thanks for the link. The Alexa Fluor 532 and 555 both seem better suited
for the laser I'm using according the the spectraviewer. A few people have
commented that the 532 also quenches quite fast though so I'll most likely
end up getting the 555.
Cheers,
Scott
>
> Have you checked out the Invitrogen spectra viewer?
> http://www.invitrogen.com/site/us/en/home/Products-and-Services/Applications/Cell-Analysis/Labeling-Chemistry/Fluorescence-SpectraViewer.html
>
> According to that you should be ok, but you will only be catching the tail
> of the dye with any practical filter set so you will suffer significant
> signal loss. You might still have enough to work with depending on
> concentration, laser power, etc.
>
> Craig
> On 2013-07-03 12:38 PM, "Scott Wong" <[log in to unmask]> wrote:
>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>> Hi everyone,
>> I was wondering if anyone has successfully used the Alexa Fluor 532
>> secondary to view tyrosine hydroxylase in dopaminergic neurons. I'll be
>> imaging it using a 543nm laser with a Nikon confocal microscope. Based
>> on
>> its
>> excitation wavelength it seems to be an ideal match but I don't any
>> experience with it.
>>
>> If not, can anyone comment on its anti-quenching properties? I'm
>> currently
>> using the Alexa Fluor 546 for the same purpose but it quenches almost
>> unusably fast with every mounting media I've tried.
>>
>> Thanks for the help,
>> Scott Wong
>>
>