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Hi Ella,

I asked the same question a while back as well (see post from April  
23rd 2007), and someone eventually did reply. Can't remember the  
specific reason why it works so well at 405 nm, but as you pointed  
out, the excitation spectrum is not quite zero at that wavelength, and  
so it's possible to kick off fluorescence and hence detect it. Not  
sure about Alexa 350, but again, I guess it depends on the excitation  
spectrum. Maybe someone else can comment on that?
On a related note, I was recently able to excite Bodipy-Cholesterol  
with a 532 nm laser and detect the red-shifted florescence with an  
EMCCD with TIRF. I was pretty surprised by that since the peak  
emission wavelength of Bodipy is around 532 nm. This just emphasizes  
the fact that excitation and emission spectra for some dyes are really  
quite broad and it is possible under certain situations to form images  
with light shifted to the end of the spectra.

John Oreopoulos



Quoting Ella Tour <[log in to unmask]>:

> Search the CONFOCAL archive at
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
> Hi,
>
> Does anybody know why DAPI can be strongly excited with 405 nm laser,
> while its theoretical excitation is almost zero at this wavelength?
> And, along these lines, has anybody tried to excite Alexa 350 dye  with
> 405 nm laser?
>
> Thank you,
> Ella Tour
>
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