Interference and fluorescence (summary)
I want to thank everyone for their expert responses on this useful
confocal microscopy application.

We have an inverted Leica microscope. Our mistake was that we either had
both the analyzer and polarizer in the light path or we removed both the
analyzer and polarizer.  In the operation of interference contrast  the
polarizer (closest to the laser light source) must be removed while the
analyzer (closest to detector) must be in the light path.

As mentioned by a number of respondents it is essential to remove the
polarizer. --Since the laser light is already polarized, the polarizer
should be removed to increase the fluorescent signal transmission in the
system.

On a second point regarding optimum resolution-- I am repeating a few
comments that are known to some investigators but unknown to other
investigators.

Owen Schwartz: "Please note that with the objective Wollaston prism in
the beam path, you will get some slight blur to the fine details in your
image.  If high resolution is needed we collect the fluorescence first
with the Wollaston removed, then put it back in the beam path and
collect the DIC image. "

Xuejun: "On another hand, for high resolution fluorescent work, I would
remove the prism from the light path. The prism does induce some
problems. You can see it easily with small beads and zoom at high level.
But for most routine work, it is acceptable."

Ken Orndorff :"Using resolution targets I have seen that the
fluorescence image is degraded slightly by the addition of the Wallaston
prism.  Generally the value of having the simultaneous morphometric
image with the confocal  fluorescence image outweighs the fuss of
collecting the images separately."


As previously reported in an E- mail communication to this confocal user
group in July 2001 --the Wollaston prism will distort point spread beads
(170nm) forming doublets. If it does it to PSF beads it will also
distort biological samples i.e. microtubules.

Thanks for your help in solving this problem?this type of communication
is very useful for the proper operation of the confocal microscope.


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]