Dear Pawel and list
I can confirm from our experience that a 488nm laser line does definitely cause some excitation of Alexa647.
We routinely do FCS with APD detectors using combinations of peptides conjugated to either Alexa488 or Alexa647 and fusion proteins tagged with either mEGFP or mKate
and when we've run controls with Alexa647 being the ONLY dye present and only the 488nm laser line, even at exceedingly low power (what we use to image Alexa488 or mEGFP with APDs), we definitely do see emission from the Alexa647 (although of course fairly minimal compared to excitation with 633nm laser).
I'm sure on any system 488nm excitation at sufficient level to excite most common green dyes would also give some cross-excitation of Alexa647 during simultaneous scanning.
Kind regards
Jen
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From: Confocal Microscopy List [mailto:[log in to unmask]] On Behalf Of Ignatius, Mike
Sent: Wednesday, 16 February 2011 06:03
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Subject: Re: cross excitation of Alexa647 wirh 488nm wavelength
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From our resident expert Dr. Iain Johnson.
"The AF647 dye itself is very stable in water, although the maleimide portion will hydrolyze rapidly to unreactive maleaimic acids in water - but dye will be fine. - I have my own data on this (measured the fluorescence quantum yield from the same aqueous stock solution on two occcasions separated by ~12 months. QYs identical within experimental error). Longer wavelegth carbocyanines (AF750 etc) are a different matter. There the dye definitely falls apart over a period of months storage in aqueous at 4C.
Using a multiband filter you give up spectral isolation in return for speed. There is really no way around this. But since it appears that they only have the 488 laser on when they do this experiment, the blame can't be entirely laid at the door of the filter. Although the excitation spectrum of AF647 looks almost at baseline at 488 nm, it never goes completely to zero on the blue side of the 0-0 band (this is true for pretty much all polyatomic fluorophores). The extinction of AF647 is about 0.3% of max at 488 nm. This is equates to about 750 cm-1 M-1 - quite sufficient to give a detectable signal when you are using laser excitation."
As communicated by Mike Ignatius, Molecular Probes/Life Technologies
-----Original Message-----
From: Confocal Microscopy List [mailto:[log in to unmask]] On Behalf Of Martin Wessendorf
Sent: Tuesday, February 15, 2011 9:22 AM
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Subject: Re: cross excitation of Alexa647 wirh 488nm wavelength
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On 2/15/2011 9:01 AM, Pawel Pasierbek wrote:
> One of our users is doing 2 color TIRF experiment, using GFP and
> Alexa647 (Alexa Fluor 647 C5 maleimid). Because the time resolution is
> very critical, we use a single filter cube with a triple filter for
> 488, 561, 633 excitation and respective emission (Zeiss HE77).
>
> Using control slide with Alexa647 we get cross excitation with the
> 488nm laser. The emission is definitely in the Alexa647 range. I have
> also looked at the sample with Confocal (LSM710) and can excite Alexa
> 647 with 488nm and even 405nm light.
>
> Has anybody observed such a phenomenon, or something happened to our
> Alexa647??? (it is 6 months old, stored at -80oC).
This sounds simply like "bleed-through" if you're using a triple filter.
The Alexa 647 will be excitable by shorter wavelengths, albeit inefficiently.
I know of no way around the problem except blocking the emission of the Alexa 647. Could you split your output to two cameras with two different emission filters? (--You would also probably need to be able to switch off GFP excitation when imaging the Alexa.)
Good luck!
Martin
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Martin Wessendorf, Ph.D. office: (612) 626-0145
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