Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

The uniform "fluorescence" you are seeing is probably reflection of a laser
off the interface between glass and sample.  If you are using an AOTF and it
is not calibrated properly, the reflection might be a from a laser different
from the one that you use for excitation.  The reflection will be worse the
more your sample refractive index differs from that of glass, which may
explain the difference between differently prepared samples.  I would verify
that there isn't a filter problem as well, by opening the pinhole wide open,
in which case your image should look very much like what you see through the
eyepieces.

Cheers,
Jeff M. Reece
Biomedical Engineer
Confocal Microscopy Center
National Institute of Environmental Health Sciences (NIEHS)
111 Alexander Drive, Bldg, 101, Rm. F219
P.O. Box 12233, MD F2-02
Research Triangle Park, NC  27709
(919) 541-0311
[log in to unmask]


> -----Original Message-----
> From: Gregory Holmes [mailto:[log in to unmask]]
> Sent: Thursday, May 12, 2005 9:54 AM
> To: [log in to unmask]
> Subject: What's wrong here?
>
>
> ---------------------- Information from the mail header
> -----------------------
> Sender:       Confocal Microscopy List <[log in to unmask]>
> Poster:       Gregory Holmes <[log in to unmask]>
> Subject:      What's wrong here?
> --------------------------------------------------------------
> -----------------
>
> 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
> 6400 Perkins Road
> Baton Rouge, LA  70808-4124
>
> P (225) 763-2520
> F (225) 763-2525
>
> [log in to unmask]
> ******************************************************
>