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Thanks for explaining this.  As a measure of bleached area it
is probably the only thing you can do but as a measure of the
actual spot size it has some limitations, most obviously that
you are measuring the spot with the same beam that created it,
so your final apparent size will be the convolution of the
spot with its bleached patch, and thus larger than the actual
spot.

Other points that come to mind:
How thin is the film and how accurate the focus?  Simple
geometry says that quite apart from diffraction effects the
beam will spread out to 242nm radius (484nm diam) at 100nm
>from focus (tan 67.5 = 2.42).  So a very small defocus (or
film thickness) will broaden the spot a lot.  If your film is
as thick as 1µm the spot will become huge.

Could there be any scattering of the beam while it is parked?
Again this would broaden the bleached area and few media are
completely free of scattering.

Chemical effects could spread the bleaching reaction but I
hope that wouldn't extend more than single-figure nanometres
(otherwise a lot of FRAP experiments would be in trouble).

                                        Guy






 Guy,
>
> To measure the spot size we use 2 techniques.
>
> 1) We make a rhodamine film on a coverslip, focus the laser on the film
>and bleach till the middle of the spot remains at twice the background.
>Then we measure the 2D intensity profile.
> 2) We park the laser in a H2B-GFP transfected cell and bleach a hole
>(again to 2 times the background). This results in a 3D laser beam profile.
> Fixed samples are also nice for doing this 3D profile.
>
> On all our systems (LSM410, LSM510NLO, LSM510Meta) we get a diameter of
>about 1 micron (FWHM) with a 63x 1.4NA lens (different lenses). It would
>be nice to see some measured values from other users, using other
>systems/brands. The diameter is rather important when doing FRAP
>experiments, so all FRAP people must be doing it.
>
> Regards, Gert