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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

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Gregory M. Holmes, Ph.D.
Director, Microscopy Core Facility

Autonomic Neuroscience and SCI Laboratory
Pennington Biomedical Research Center
6400 Perkins Road
Baton Rouge, LA  70808-4124

P (225) 763-2520
F (225) 763-2525

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