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Thanks for all the information...  Much appreciated!

Regards,
Jason

----------------------------------------------------------
Dr Jason Goh
School of Electrical & Electronic Eng.
University of Nottingham
TEL:  +44 (0) 115 951 5556
FAX:  +44 (0) 115 951 5616

>>> [log in to unmask] 11/18/03 01:44pm >>>
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Just to add a bit to Nuno's reply, if you look at the FITC
spectrum at the site he gives you will see it has two peaks,
one at ~790nm and the other at ~930nm.  What this tells us is
that the shorter wavelength peak is exciting to a higher
electronic state which automatically means that the risk of
bleaching is increased.  And in practice many people find that
FITC bleaches very rapidly indeed at these wavelengths.  It
is much more stable at 930nm.

Why is it not 988nm?  Because fluorescein is a very symmetrical
molecule and selection rules say that for a symmetrical molecule
2P excitation cannot occur to the same state as 1P.  If you look
at the spectra for asymmetric molecules such as DAPI you will
find that they are much closer to the 1P spectra at twice the
wavelength.

                                           Guy Cox



Quoting Nuno Moreno <[log in to unmask]>:

> Search the CONFOCAL archive at
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal 
>
> Dear Janson,
>
> The virtual state in the 2P absorption is not so spectrally defined. For
>   FITC you can have a good signal starting at 790 nm. The absorption
> spectrum dependents a lot with the fluorofore and its much more wide
> than 1P.
>
> Here you can find some 2P absorption spectra
>
> http://microscopy.bio- 
rad.com/products/multiphoton/Radiance2100MP/mpspectra.htm
>
> All the best,
> Nuno Moreno
>
> Jason Goh wrote:
>
> > Search the CONFOCAL archive at
> > http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal 
> >
> > Hi all,
> >
> > At the moment I'm designing a variant on a two-photon fluorescence
> microscope and as an optical engineer, have a question on a practical aspect
> of the operation.
> >
> > I'll be using fluorescein (FITC - excitation peak 494nm) as my fluorophore
> which would suggest a two-photon excitation wavelength of 988nm.  In the
> literature, I've not seen anyone use an excitation wavelength this high -
> mainly seems to be <800nm.  Is there any practical reason for this?  Is it
> due to decreased laser efficiency outside the optimum running wavelength
> (approx. 800nm).  I would have thought it was always best to operate at the
> excitation peak of the fluorophore to get more emission for the same amount
> of incident power...
> >
> > Thanks in advance for your help,
> > Jason
> >
> > ----------------------------------------------------------
> > Dr Jason Goh
> > School of Electrical & Electronic Eng.
> > University of Nottingham
> > TEL:  +44 (0) 115 951 5556
> > FAX:  +44 (0) 115 951 5616
> >
>
> --
> ___________________________
> Nuno Moreno
> Phone: +351 214464606
> Fax    : +351 214407970
> www.igc.gulbenkian.pt 
> Cell Imaging Unit
> Instituto Gulbenkian de Ciência
> Portugal
> ___________________________
>


--
Associate Professor Guy Cox
Electron Microscope Unit, F09
University of Sydney NSW 2006
+61 2 9351 3176