Re: Strange artifact in confocal Z-stack

Posted by Monique Vasseur on
URL: http://confocal-microscopy-list.275.s1.nabble.com/Strange-artifact-in-confocal-Z-stack-tp593216p593229.html

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Dear all,

I just want to complete my first response saying that we could see the same pattern in bright field on a Nikon upright fluorescence microscope when we used a empty position in the widefield fluorescence filter cube turret.

When in confocal mode, no widefield fluorescence filter cube should be in place.  When we were looking in brigtfield that day, we also were without fluorescence filter cube in place but the mercury lamp was on and the mercury lamp shutter was not closed. As soon as we closed the fluorescence shutter, the pattern disappeared in brightfield.  That was how we found that we needed an empty cube in that position to avoid interference with the mercury lamp light reflection/leak in the light path.

Monique Vasseur
Microscopie et imagerie
Département de biochimie
Université de Montréal
C.P. 6128, succursale Centre-ville
Montréal QC    H3C 3J7   Canada
tél. [phone] poste 5148
-----Message d'origine-----
De : Confocal Microscopy List [mailto:[hidden email]] De la part de Stephen Cody
Envoyé : 2 juin 2008 20:07
À : [hidden email]
Objet : Re: Strange artifact in confocal Z-stack

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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
PO Box 2008 Royal Melbourne Hospital
Victoria,      3050
Australia
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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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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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