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Greg,

Only a guess, but one of the things that can be happening is that you are
getting some sort of delicate autofluorescence from the substrate (hence
the full field fluorescence at that particular plane) and that the cell
edge is acting like a lens to focus that illumination.  If there is an
aperture iris on the fluorescence illuminator, try adjusting it to see what
happens.

Let me know your results.

Barbara Foster
Microscopy/Microscopy Education
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Piel.

At 08:53 AM 5/12/2005, Gregory Holmes wrote:
>Search the CONFOCAL archive at
>http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
>All -
>
>Since I am mainly a sectioned tissue microscopist just learning to deal
>with imaging single layer cultured cells, maybe you've heard of this one
>before...
>
>A colleague has cultured cells (raised on glass coverslips treated with
>polylysine). They have been staining for a particular receptor (Cy3
>secondary antibody before, and Cy2 secondary after treatment) and want
>to look at changes in receptor density at the membrane surface.
>
>Under widefield fluorescence one sees what appears to be an increase in
>fluorescence at the membrane boundary (picture the cell as having a band
>of fluorescence forming it's border). This phenomenon appears to occur
>close to the coverslip surface.
>
>Under confocal at the same focal plane, all that we're detecting is a
>near uniform field of fluorescence (as if we're trying to image the
>coverglass) espescially with the 488 laser line. No cells are
>distinguishable. I've checked the pinhole & collimator settings. I even
>tried to decrease the optical thickness in order to not impinge on the
>coverglass. Nothing... We go from a full screen of "light" to the top
>edge of the cell (I'm thinking they're only about 3-5 microns big
>judging from how few z-sections were getting at 0.4 micron optical
>thickness).
>
>Images of my tissue (dual fluorescent immunolabeling in apposition to
>eGFP tagged pseudorabies virus in spinal cord cells) come out
>beautifully.
>
>Since when can a widefield generate a better image than a confocal???
>I'm suspecting that the widefield is generating some false image, but
>since it's producing what they want to see it's a matter of "let's
>publish the widefield data rather than that cruddy confocal data..."
>
>I've never had confocal images look so crummy.
>
>Thoughts?
>
>Greg Holmes
>
>******************************************************
>Gregory M. Holmes, Ph.D.
>Director, Microscopy Core Facility
>
>Autonomic Neuroscience and SCI Laboratory
>Pennington Biomedical Research Center
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>
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