Considering the principles of the optics, it makes sense to me that you
would have
seen a doublet.  The wollaston prism, in principle sets up 2 separate light
paths
with one being spatially separated from the other by a few tenths of a
micron
(my understanding).  In principle, perhaps there is a way to split off the
signal so that the "fluorescence" path is fed through it's own set of
polarizers.  You can determine, theoretically, the directions of
polarization of the 2 separated beams.  They are 90 degrees separated from
each other in polarization, you can then use a polarizer between the
objective and the camera in order to block out one of the 2 beams.  This
would
leave just one beam-path.  The down-side, of course, is that you will give
up significant
signal, because you have to first split the beam off (signal/2) and then
block one of the
2 polarized beams (another signal/2 loss).

-----Original Message-----
From: Confocal Microscopy List
[mailto:[log in to unmask]]On Behalf Of Robert Zucker
Sent: Wednesday, July 25, 2001 4:45 PM
To: [log in to unmask]
Subject: Eggs or balls.


This is an Interesting observation with a question for the confocal user
group

I was trying to do Huygens deconvolution ( Bitplane software)  to increase
the resolution of some confocal images. In order to do this effectively you
are supposed to use 0.17 micron beads ( balls) or 0.5 micron beads( balls)
with high power objectives to get a reference particle for the software.
While doing this test to acquire the necessary reference images from  the
0.17 u beads, they appeared as doublets( two beads) and the 0.5 micron
beads appeared egg shaped. What will this type of distortion observed on
beads do to the fluorescent images of cells and particles?

 On closer inspection we notice the Wallistron prism, which is necessary
for interference contrast, was in the light path. I interpret these results
to mean that while doing interference contrast simultaneously with
fluorescence there will be a degradation of the fluorescence signal.  Does
any one know by how much? Should we not use interference contrast when
acquiring  high resolution fluorescent confocal images?
Although interference contrast is a useful signal, it should not degrade
the fluorescence signal if ones wants optimum resolution from a specimen. I
would like to have some input on this observation and any suggestions or
recomendations on the simultaneous use of both signals. Thanks
Bob

Robert M. Zucker, PhD
U.S. Environmental Protection Agency
MD 72
National Health and Environmental Effects Research Laboratory
Research Triangle Park, North Carolina, 27711
Tel: 919-541-1585; fax 919-541-4017
e-mail: [log in to unmask]