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Afternoon Elaine,

We've are beginning to use reflection to sort out z-displacement of
reflecting inclusions in almost transparent crystals.  The issue is to
'find' the depth of the most distant, last and thinnest optical section, and
this doesn't work well if you are looking at a stack of reflectors.  In your
case, it is a question of the thinness of the optical section and the depths
of the pits.  I know I've reached the back side of a pit when my PMT says,
"Ouch!", or the image of the inclusion loses most of its intensity.  We have
just begun to work up this method, so there are no data to report on what
objective works with what size inclusion.  The catch-22, of course, is that
the thinnest optical sections come through the smallest apertures, and for
the most critical work we really should be using an upright microscope with
the aperture at the true back focal plane of the objective.[I think!]  For
us, the key nuance was realizing that to do this job well, we didn't want a
big stack, we wanted a statistical stack of very thin sections.  The rest of
it involves the minimum precision we can accept for this task.

To calculate volume, you need to know depth and have an accurate sigma of
areas, and I'm only thinking of a pit with vertical walls.  If there is a
pit with greater diameter at its base, then I would bet that at some depths
the reflections from the wide base could cause what might be considered to
be a flare of greater diameter than the pit opening to be acquired at the
level of that opening within the thickness of the optical section.

[Oops, now I've gone and closed the closet door, and I'm seeing visions of
confusing ray diagrams and dx's over dy's, and lambda's, and I've got to
stop.]

[Gosh almighty!  When we learned microscopy we thought that seeing the
difference between liver and kidney was cool.  Then we had to learn physical
chemistry so we could understand what the kidney was doing.  Now we have to
learn wavelet analysis and quantum mechanics so we can determine the depth
of a hole.  And if the epiglottis is open, what drives the bad consequence
of a pneumothorax if it isn't good old microanatomy?]

Hope what came before "Oops!" and "Gosh!" helps,

Fred Monson

Frederick C. Monson, PhD
Center for Advanced Scientific Imaging
Schmucker II Science Center
West Chester University
South Church Street and Rosedale
West Chester, Pennsylvania, USA, 19383
Phone:  610-738-0437
FAX:  610-738-0437
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CASI URL:  http://darwin.wcupa.edu/casi/
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> ----------
> From:         Elaine Kunze
> Reply To:     Confocal Microscopy List
> Sent:         Thursday, June 6, 2002 11:39 AM
> To:   [log in to unmask]
> Subject:      Metal pits
>
> Has anyone ever used confocal microscopy to measure the volume of pits in
> a metal surface?  I have tried reflective measurements, but can't tell
> where the pit begins or ends.  I know its a long shot but I figure if
> anyone knows, it will be this group.
>
>
> **************************************************************************
> **
>         Elaine Kunze
>         Flow Cytometry.....Image Analysis...
>         Life Sciences Consortium
>          http://www.lsc.psu.edu/stf/cqca/home.html
>         8B Althouse Laboratory  (814-863-2762)
>         Penn State University
>         University Park, PA 16802
>
>