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Ok, now I got it. Fortunately, we have different dicroics so I will exchange them and check for
changes on the PSF.

I analyze the aberrations not directly on the plot of the PSF instead I analyze the changes of the
2D fwhm over z (separated for x and y direction), which e.g. leads to the graphs on the left in the
attached image. There is an offset between the x and y fwhm and both curves also seem a little
shifted over z.

Choosing a particle that is not centered (here I choose one that is on the outer right of the field
of view but same y coordinate) changes the offset/shifting and in this example even leads to a
perfect match of the curves (graph on the right). Maybe this also points to a specific aberration?

I recognized that attaching files is not allowed on that list so I will send the image directly.

Best,
Stefan

Sebastian Rhode wrote:
> > *****
> > To join, leave or search the confocal microscopy listserv, go to:
> > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> > *****
> >
> > Hi Stefan,
> >
> > I might be wrong in your case, but I have seen quite a lot of systems, where
> > a rather thin dichroic introduces optical problems. Imagine the dichroic is
> > bend a bit due to its mounting along on axis. So this can lead to a curved
> > surface along one axis of the dichroic. But this curvature introduces a
> > focal length along on axis.
> >
> > And finally those kind of things will "mess up" your otherwise perfect PSF.
> > To avoid we use 2mm substrates (TITF, FRAP, WF) inside the microscope or
> > even 5mm substrates for our dichroics inside our spinning disc system.
> >
> > Cheers,  Sebi
> >

Stefan,

Would you be able to post to the web somewhere a few example images so we can see as well? Maybe
even a XZ and XY cross-section reconstructions through a few beads? Is the effect the same all
across the field of view? The shape of the PSF can sometimes tell you if there are any specific
aberrations in play.

The best way to isolate the problem would be to move the objective over to another scope (without a
CSU) and try collecting the same data on the same type of sample. What happens if you use a similar
objective on the original scope system? Then you could also try removing the CSU on your first scope
and connect the camera directly to the upright port, and again collect the data with the objective
in question (use the arc lamp for a light source and scope filter cube). This will determine if it's
the objective, the scope, or maybe the CSU.

Cheers,

John Oreopoulos
Research Assistant
Spectral Applied Research
Richmond Hill, Ontario
Canada
www.spectral.ca
-- 
---------
Dipl. Ing.-Inf. Stefan Sokoll
Lab. Molecular Physiology
Dept. Neurochemistry & Molecular Biology
Leibniz Institute for Neurobiology (LIN)
Brenneckestrasse 6
39118 Magdeburg
Germany

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