Olympus FV1000 image problem

[Jacky Liang]

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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

[Vladimir Ghukasyan-2]

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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 <[hidden email]> 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
>