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Neal,

We did confocal of 250 micron thick liver slices for before & after
toxicology experiments a few years ago.  We were using the BiopTechs Live
cell Delta T dishes and live/dead dyes from Molecular Probes.  The confocal
we originally performed this on was an inverted microscope and we
discovered several problems with this configuration.

1) the surface we were imaging was resting on the bottom (glass) surface of
the dish.  This worried us as far as keeping the cells on that side of the
slice "happy" over time.  Without the ability to exchange nutrients and
oxygen with the culture media,  would the cells die faster due to the poor
environment, rather than the toxic exposure?

2) were we exposing the bottom of the slice to the same levels of toxic
substances as the top?  Same question re: penetration and exposure to the dyes.

3) dye penetration seemed to only be 2-3 cells deep, probably partly due to
light scattering and partly due to dye penetration.  Multiphoton may have
improved these results, but we didn't have one at the time.

4) we tried suspending the slice, using the BiopTechs brain slice
adapter.  With the peristaltic pump on, the slice "flapped in the current",
which was not conducive to good imaging.  Also, the slices hang in an arc
between the firmly attached edges.  This, combined with the limited
penetration of the live/dead dyes, meant that we tended to image
fluorescent "doughnuts" as we optically sectioned into the slice.

5) another interesting anomaly, our slices were usually roller cultured
before we used them.  My impression after imaging a bunch of slices was
that cells tended to wash off in the areas farther away from the
vasculature and were a bit more firmly attached  in the areas around the
vasculature (where there is more connective tissue).  Again, optical
sectioning (& 3D rendering) tended to give the impression of mountains and
valleys, instead of a relatively smooth surface.

As a result of this experience, when we had opportunity to purchase a new
confocal we went with an upright and "dipping" lenses.  I realize that
there are some compromises that this configuration brings, but for what our
toxicology group wanted to do, this seemed like the best choice.

Doug

At 11:04 AM 2/8/2005, you wrote:
>Search the CONFOCAL archive at
>http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
>Hi all,
>I have a user that is interested in doing fluorescence microscopy of
>living hepatic tissue slices.  I have not heard of anyone doing anything
>similar and I wondered if anyone in the group has had any experience
>doing anything similar and might have protocols or references.
>
>Thanks,
>
>Neal R Gliksman, Ph.D.
>Senior Application Scientist - Screening
>Meta Imaging Products
>Molecular Devices Corporation
>402 Boot Road, Downingtown PA 19335
>Ph: 610 873 5610
>
>http://www.moleculardevices.com/

.....................................................................
  Douglas W. Cromey, M.S.          Dept. of Cell Biology & Anatomy
  Research Specialist, Principal   University of Arizona
  (office:  AHSC 4212A)            P.O. Box 245044
  (voice:  520-626-2824)           Tucson, AZ  85724-5044   USA
  (FAX:  520-626-2097)             (email: [log in to unmask])
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