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For clarification:

This artifact occurs in a single channel exploration only, something
that I didn't mention. My thoughts were that this problem has
something to do with reflection of emission light from somewhere in
the sample preparation (between slide and coverslip) as the noise
seemed to be stable and dependant on the depth of focus. The tissue
is slightly irregular and perhaps as much as 1 mm thick. The
placement of the coverslip is not necessarily uniform from one slide
to the next.

Has anyone seen this sort of artifact on any system or with any
sample preparation? Any comments addressing the theoretical are as
welcome as those addressing the more pragmatic side of things. Thanks
in advance, Cameron

>Search the CONFOCAL archive at
>http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
>Dear List,
>In collection of a confocal Z-stack of images, a certain artifact
>appears in about 10 middle planes of a 40 plane stack. As the depth
>of focus changes for these 10 planes, a "front' of very intense noise
>sweeps across the region of interest, covering it completely for 2 or
>3 planes, rendering the data of this stack unusable. Does anyone have
>experience with this sort of problem? Could anyone share their
>insights into what causes this and/or advice about how to prevent
>this it the future?
>The instrument is a Bio-Rad Radiance 2000 confocal unit connected to
>an Olympus IX-70 inverted microscope. The magnification is 20x UPlan,
>NA = 0.07. The sample is a thin whole mount, between slide and
>coverslip, of a tissue probed with green fluorescent beads. The depth
>of the stack is 40 microns with step size of 1 micron. Thanks, Cameron
>--
>Cameron Cooper
>Molecular Cytology Core Facility
>Molecular Biology Program
>2 Tucker Hall
>University of Missouri
>Columbia, MO 65211-7400
>
>email: [log in to unmask]
>(573) 882-4895 (lab)
>(573) 882-0123 (fax)
>Web page: http://www.biotech.missouri.edu/mcc/