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June 2014

CONFOCALMICROSCOPY@LISTS.UMN.EDU

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From:
Jacky Liang <[log in to unmask]>
Reply To:
Confocal Microscopy List <[log in to unmask]>
Date:
Thu, 12 Jun 2014 15:20:22 -0500
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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

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