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-tp593216p593230.html

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We had something similar with a Nikon upright microscope, except that we could see a similar effect in brightfield images -when we were using the "without fluorescence filter" position of the filter cube turet.  This was due to a leak of light in the filter cube turet when there was nothing in the "without fluorescent filter" position.  As soon as we put an empty cube in place, no more halo appeared.

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 Christophe Leterrier
Envoyé : 29 mai 2008 04:30
À : [hidden email]
Objet : 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