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Hi, I agree with Barbara and Xuejun comments on the first problem, it might
be either one or could be both responsible for that.

For the second problem, please check the laser power, because, we usually
get horizontal lines when the laser's anode current increased as well as
when the laser power drops considerably. For the spots, try different
objectives first and see you get the same number and localization of the
spots in all images. If you do get the spots irrespective of the objective
used, rotate the scan head while you are using a high magnification and see
the spot/s is/are also rotating. If you do see the spots are also rotating,
then the confocal head needs to be cleaned. If you don't then under the
objective and below the filter wheel, there would be a magnifier lens which
enable you to switch (push or pull) between 1 or 1.5 magnification (this
depends on what microscope hooked up with your confocal system, however),
try to clean this carefully using a pressurized clean air or with a cotton
swab dipped in diluted Sparkle (glass cleaning liquid without ammonia).
Usually, it is hard to reach that area/surface.

If you could send me an image offline, I will try to recognize how far they
look alike compared to our images, when there was a problem of same kind.

Shiv

Mayandi Sivaguru, Ph.D.,
Associate Director
Molecular Cytology Core Facility
Molecular Biology Program
2, Tucker Hall
University of Missouri
Columbia, MO 65211-7400
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Voice: 1-573-882-4895
Fax: 1-573-882-0123

www.biotech.missouri.edu/mcc/




At 11:28 AM 4/3/03 -0700, you wrote:
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> >===== Original Message From Confocal Microscopy List
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> >Search the CONFOCAL archive at
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> >
> >Dear All,
> >
> >We have a Zeiss LSM 510 setup, version 2.5. We recently acquired a 100x
> >objective (plan-neofluar 100x/1.3 oil). We are unable to do Z sectioning
> >using this objective, and always get the same error message
> >"Range exceeds limit of 1um for plan-neofluar 100x/1.3 objective. Reduce
> >slices and interval"
> >If I reduce the stack size to less then 1 um then it will do the z section,
> >but obviously this is not much use.
> >What is going on?
> >
> >A second problem we have with both the 40x and 100x oil objectives, is that
> >the transmission DIC images are full of the black spots and streaks, which
> >are consistent every time we scan. The Zeiss technician has taken apart and
> >cleaned everything he can in the beam path, but there is no improvement. Any
> >suggestions will be gratefully received.
> >
> >Thanks,
> >
> > Rebecca Haffner, Ph.D
> > Dept. of Veterinary Resources,
> > Weizmann Institute,
> > Rehovot, Israel.
> > 972 8 9342719 (fax 972 8 9342823)
> > [log in to unmask]
>
>Hi,
>
>The 1st problem is related to Zeiss's "smart" LSM system. the LSM software
>will not allow you to obtain z-stack thicker than the working distance of the
>lens you are using. In this case, the software "thinks" the lens has a working
>distance of 1um. I think somewhere the database for objectives is messed up.
>go to "maintenance", "objective" and I do not remember if you can edit the
>database there. But you can certainly create a new objective with whatever the
>working distance it should be there, you need to invent a objective number
>though. then you will be fine. If you have trouble to get it done, email me
>and I will give you the detailed procedute how to do it (I am not at work
>now). Or you can ask your local Zeiss guy to edit the database and put the
>correct value for the lens in it (use "dbtool" open your LSM database, under
>available or potential objectives).
>
>As to the DIC images, I am sure you thought of these already, set up the
>Kolher illumination and set the iris on condensor  wide open. See if this
>helps. Another thing to check if your immersion oil has crystals in it.
>
>Good luck!
>
>Xuejun