>My institution has just acquired a Zeiss LSM 510 equipped with a
>Coherent Innova Enterprise UV laser, which I thought of using for
>DNA staining with DAPI or Hoechst 33342. I immediately ran into
>a very annoying problem, which I suspect might be a consequence of
>being a total CLSM newbie:
>
>The images I get with either the 63x Apochromat or the 100x
>Apochromat are totally "unevenly illuminated", very bright in the
>central part and dim towards the margins. This is most pronounced
>when I select the largest scanning fields.

I have NO specific experience with Zeiss 510 uv systems.  The
following comments are from basic principles, but they may help.

The problem is clearly what photographers cause 'vignetting' - ie
at large scan amplitudes "something" is obstructing the beam.  It
is not in a plane conjugate with the sample or you would see a sharp
edge.  One possibility (but not the only one) is that the beam is
not pivoting accurately about the entry pupil (back focal plane)
of the objective.  Since the ultraviolet light-path has compensating
(beam shaping) optics in it to correct for the fact that the lens
is not achromatic in the uv it is quite possible for the uv to
be misaligned (or rather, mis-focussed) while the visible light
is fine.  To check on this possibility first get yourself a pair
of proper laser safety u/v absorbing glasses - DON'T trust the
orange shield on the microscope or any other makeshift.  Remove
a lens and place a piece of fluorescent paper (ordinary white
paper is usually fine) on the stage.  The circular patch of
fluorescence should be stationary.  (In principle it should
be stationary with the piece of fluorescent paper over the hole
where the lens was but with infinity corrected optics it should
still be static - but larger and fuzzier - at the stage).
If not, you have located your problem but I've no idea how you
fix it.  There must be an adjustment for the purpose.

                                                Guy Cox

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