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Here are two more references which go back a few years but are quite good.
N.S. White et. al.
"Aberration control in quantitave imaging of botanical specimens by
multidimensional fluorescence microscopy"
Journal of Microscopy Vol 181, Pt. 2, February 1996 pp. 99-116 (Royal
Microscopy Society)
" Multidimensional Fluorescence Microscopy: Optical Distortions in
Quantitative Imaging of Biological Specimens"
Fluorescent Microscopy and Fluorescent Probes, Plenum Press, New York,
1996 pp. 47-56
Regards,
Tom
Tom Donnelly Applied Precision, LLC
Biotechnology Group 1040 12th Ave. N.W.
(425)657-1294 Issaquah, WA 98027-8929
(425)557-1055 fax [log in to unmask] http://www.api.com/lifescience/spectris.html
-----Original Message-----
From: Stefan Gunnarsson [mailto:[log in to unmask]]
Sent: Tuesday, December 02, 2003 1:41 AM
To: [log in to unmask]
Subject: RI mismatch?
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Hi!
We have been looking at rather thick sections of tissue, cut at 30-35
microns, stained with propidium iodide, and mounted with DABCO mount
(which I assume has RI around glycerol).
With a 20x/0.7NA Imm lens with glycerol as immersion medium, the
z-distance between top and bottom of the section (as measured by the
fine focus device on the microscope) was the expected 30-35 microns.
With a 40x/0.7NA dry lens, however, the same distance was only around
20 microns.
The decrease in signal intensity away from the cover slip was slight in
both cases, but a little bit more pronounced with the 40x lens.
My immediate thought was that this was an example on RI mismatch, but
the clever student objected, since the distance was actually measured
with the step motor on the microscope. ("A stick in the lake will only
seem to be bent, if actually measured with a meter the true distance to
the surface will be revealed").
Could someone please shed some light on this....
tia,
Stefan
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Dr Stefan Gunnarsson
Evolutionsbiologiskt Centrum Evolutionary Biology Centre
Enheten för biologisk strukturanalys Microscopy and Imaging Unit
Norbyvägen 18A
SE75236 Uppsala, Sweden Tel & Fax: +46 - 18471 2638
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