Re: Strange artifact in confocal Z-stack

Posted by Stephen Cody on
URL: http://confocal-microscopy-list.275.s1.nabble.com/Strange-artifact-in-confocal-Z-stack-tp593216p593228.html

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D'day Christophe,

I think that it is non-specific fluorescence on the outside of your tissue section. Did you use an antibody label? If so it may represent non-specific binding to the outside of the section. If it was a lipophilic retrograde label such as DiI, these can wash out of the membrane when in glycerol based mountants, and it may have become trapped between the section and the coverglass.

The curved nature of the artefact is probably as Paul suggests, because of the use of a non plan objective. Further evidence that you may be looking at the outside of the tissue section is that there appears to be knife chatter marks visible in panel 5.

Cheers

Stephen H. Cody
Microscopy Manager
Central Resource for Advanced Microscopy
Ludwig Institute for Cancer Research
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-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Christophe Leterrier
Sent: Thursday, 29 May 2008 6:30 PM
To: [hidden email]
Subject: Strange artifact in confocal Z-stack

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http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

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


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