Hi Andreas

We published an actual bleach experiment here:

Cannell, M.B. & Soeller, C. (1997) Optical sectioning in fluorescence 
microscopy by confocal and 2-photon imaging techniques. Microscopy Today 
5(8):12-15.

You can see that  bleaching during scanning in wide field is the same 
throughout the sample (unlike 2P). This is because the 'dose' is the 
same at all points.

Cheers Mark


Bruckbauer wrote:
> Many multiphoton talks and reviews show an image where a laser beam is
> focused into a fluorescent solution. This is done to demonstrate the
> localized nature of 2Photon excitation compared to the 1-photon case, see
> the W.R. Zipfel W.W Webb review in Nature biotechnology 2003, 21, 1369.
> Usually low NA, long working distance lenses are used. 
> What puzzels me is that, when calculating the intensity distribution for the
> one photon case, e.g. with the Huygens Nynquist calculator set to wide field
> mode, i get a focused spot too. The distribution has wings which are not
> present in the 2 Photon case, but the effect should not be so dramatic as in
> the images typically shown. After all it is "only" a quadratic vs. a linear
> relation. 
> I think that when doing microscopy and scanning a laser beam through the
> sample, things can be different, the wings can have a big effect in
> bleaching the sample. However i find the image shown a bit misleading. Has
> anyone done the experiment and can tell me how this works?
>  
>