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>Search the CONFOCAL archive at
>http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal 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
>


Hi Gert,

This seems an interesting method.

However, it might yield quite a different result from conventional methods.

This would occur if, as seems quite likely, at least some bleaching
is non-linear (i.e., the RATE of bleaching, measured in dead
molecules per excitation, may depend on the intensity of the light;
perhaps varying with the square of the intensity).

Under these circumstances, with your method the bleaching at the
center of the spot would occur more rapidly than if the bleaching
were linearly proportional to light level. This would tend to make
the distribution narrower (looks like higher) resolution. If
bleaching were a square-law process, this would reduce the width of
the bleached spot by about 1.4x.

And don't forget, if you could image the PSF as an Airy disk with
rings around it, the Rayleigh-criterion resolution is HALF of the
diameter of the first dark ring.

Still, it is a test that is easy to do and will give a useful
benchmark.  Good idea.

Jim P.
--
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Prof. James B. Pawley,                             Ph.  608-263-3147
Room 223, Zoology Research Building,               FAX  608-265-5315
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"A scientist is not one who can answer questions but one who can
question answers."  Theodore Schick Jr., Skeptical Enquirer, 21-2:39