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Jacky,

This kind of effect usually indicates that the stage insert is not aligned
properly. Put a thin specimen in the insert and adjust the alignment
screws. In your case you clearly have a N-S tilt. If you can, try also an
insert from another system.

Best wishes,
Vladimir



On Thu, Jun 12, 2014 at 4:37 PM, Jacky Liang <[log in to unmask]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> Hello,
>
> When scanning close to the top or bottom of the tissue (sagittal mouse
> brain
> in our case), our FV1000 is showing uneven illumination (very obvious
> brightness gradient) this image (dropbox link) shows the problem,
> https://www.dropbox.com/s/1g53fj03n5fafev/img1.jpg
> it's a mosaic of 35 images (20x) stitched with Fluoview multiple time lapse
> function.
> basically, when looking at the top or bottom of the tissue, half of the
> image will get darker. The problem is more visible on lower magnification
> (10x and 20x), but visible on 40x too.
>
> it doesn't seem to be a problem related to uneven thickness of the specimen
> : 1) same problem is present using a 'flat' specimen from the Olympus tech;
> 2) no matter how we adjust the specimen holder, the problem persisted; 3)
> the problem is not present on a different confocal system using the same
> sample.
>
> the Olympus tech had spent over 5 days in total trying to find/fix the
> problem, he re-aligned/adjusted the system twice (before and after changing
> a new optical cable), no luck...
>
> any input is appreciated
> Thank you
> Jacky Liang
>