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Hi all...

I've been trying to determine the best way to correct
for (and understand) axial distortion in a thick specimen (greater than 50
um) composed of a material of unknown refractive index.  I am using a 20x
objective (NA=0.75) on a Zeiss 510 LSM.  If someone could comment on the
following, I would be most appreciative:

Axial distortion can be attributed to:

1.  Refractive index mismatch between immersion (air in this case) and
mounting medium (a glycerin-based gelatin, RI = 1.57)
2.  Point spread function broadening
3.  Refractive index mismatch between the sample itself and the mounting
medium

I have imaged 3.4 um fluorescent beads under the same mounting and imaging
conditions as the sample and find that I need to use a correction factor
of ~0.5 to obtain a near spherical reconstruction of the beads.  I have a
paper (Visser and Oud, 1994, Scanning 16:198-200) which states that axial
distortion can be corrected for by simply determining the ratio of the
lateral and axial sizes of the sphere and dividing all vertical dimensions
by this elongation factor.  I've read several other papers on axial
distortion, but most deal with oil immersion rather than air lenses,
and I am not sure if they apply to my particular conditions.

My general question is:  am I approaching this problem in a reasonable way
without knowing the refractive index of the material composing the
specimen itself?  My background is primarily in electron optics, so I
apologize in advance if my understanding of light optics is a bit sketchy.

Any comments would be greatly appreciated.

All best,

Angela
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Angela V. Klaus, Ph.D.
Director, Microscopy and Imaging Facility
American Museum of Natural History
Central Park West at 79th Street
New York, NY 10024 USA
Email: [log in to unmask]
Tel: 212-769-5977
Fax: 212-496-3480
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