Re: Strange artifact in confocal Z-stack

Posted by Guy Cox on
URL: http://confocal-microscopy-list.275.s1.nabble.com/Strange-artifact-in-confocal-Z-stack-tp593216p593231.html

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I'm sure it must be a reflection from something - either slide,
coverslip or an air bubble in the sample.  2 suggestions to
check this out:

1. move the spectral detection sliders further from the excitation
wavelength and see if that makes it fainter.
2.  try an XZ section which might reveal the source.

By the way, thanks for putting the pictures up - it's so
much easier to comment on pictures than a vague description!

                                                 Guy



Optical Imaging Techniques in Cell Biology
by Guy Cox    CRC Press / Taylor & Francis
    http://www.guycox.com/optical.htm
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Associate Professor Guy Cox, MA, DPhil(Oxon)
Electron Microscope Unit, Madsen Building F09,
University of Sydney, NSW 2006
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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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