Re: Strange artifact in confocal Z-stack

Posted by Sarah Kefayati on
URL: http://confocal-microscopy-list.275.s1.nabble.com/Strange-artifact-in-confocal-Z-stack-tp593216p593227.html

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Dear Christophe,

 

I would say it is the reflection of your coverglass as I have seen this artifact once when I used an unknown coverglass.

do the imaging with the different cover glass and check if it's still there. 

 

Sarah

On Thu, May 29, 2008 at 8:50 PM, Paul Rigby <[hidden email]> wrote:

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Christophe,
What objective was used? We often see a similar reflection pattern from the coverslip or slide when using non-Plan objectives that have a curved focal plane.

We also sometimes see a pattern like this when a small air bubble is present in the immersion or mounting medium. And the bubble always migrates to the exact centre of the field!

Guy's suggestions should rule out reflections as the source.

Cheers
Paul Rigby


Dr Paul Rigby
Senior Lecturer
Centre for Microscopy, Characterisation and Analysis (M510)
The University of Western Australia
35 Stirling Highway
Crawley  WA  6009
Ph (61-8) 9346 2819
Fx (61-8) 9346 3469

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Christophe Leterrier
Sent: Thursday, 29 May 2008 4:30 PM
To: [hidden email]
Subject: Strange artifact in confocal Z-stack

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Dear all,

One of my colleagues brought me a Z-stack today that has a circular
pattern superimposed on his sample. It is taken with a Leica SPE
confocal, pinhole at 1 Airy unit, I guess the Z step is about 0.2 µm.
If you go down the stack trough the cell toward the glass (cells on
glass coverslip observed on an upright microscope), you see a circular
ring halo appearing that lowers in radius up to a circular spot at the
center of the field. A picture is worth a thousand words, so please
check the problem here :

http://ftp.espci.fr/incoming/christo/Montage.jpg

I've seen reflections from the coverslip before as a high background
fluorescence appearing when you reach the glass surface, but I wonder
if this is reflection or a defect in the objective, or something else.
Sis anyone have a similar problem ?

Thanks a lot for your help,

Christophe



Christophe Leterrier

Postdoc
INSERM UMR641 Neurobiology of ionic channels
IFR Jean Roche - Mediterranee University
Marseille, France