With reference to some previous discussions on this group regarding the
elongation of confocally-imaged objects along the Z-axis, I had a few questions
about the extent of elongation actually observed due to differences in
refractive indices.
 
We have done some preliminary studies with 4, 10 and 15 micron beads labelled
with Fluorescein, using a 40X oil objective.  The beads were embedded in
100 micron slices of gelatin. The slices were mounted either in water,
immersion oil,  methyl salicylate or GelMount (Biomeda Corp).  A Biorad MRC500
was used for the imaging, with a Z-axis stepper motor controller.
 
We did *not* observe elongations to the degree predicted by theory (Visser et
al. 1992, Optik, vol. 90) for small beads, and even lesser elongation for the
bigger beads.  In the case of water, an elongation of upto 2.5 times the XY
dimension is predicted, the factor reducing as the r.i. of the mounting medium
 approaches that of oil.
 
Has anyone else observed elongations of upto 2.5 times the XY dimensions of
the beads, in the case of water for example?
 
If anyone has ever measured the refractive index of GelMount, would it be
possible for you to discuss how you measured it, and what your conclusions
were?
 
 
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Y.S. Prakash
4-176 Anesthesia Research. SMH                  ph: 507-255-7328
Mayo Clinic
Rochester, MN55905
 
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