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Dear Stanislav.


You might have a look at

http://micro.magnet.fsu.edu/primer/techniques/dic/dichome.html

Depending on what your concept of "theoretical" is, this might be the
source of information you are looking for - or not.

The wording "behind" the objective in your text is a little bit un-clear.
What you mean, I suppose, is the following:

In a classical transmitted light configuration, you will have the light
source, a collector lens, the field diaphragm, then the DIC polarizer, the
condenser DIC prism - also known as the "main DIC prism" -, the condenser
with its front lens, the condenser immersion medium (air, water, oil, or
glycerine),  the microscope slide, the specimen in its mounting medium,
the cover slip, the immersion medium for the objective - e.g. air, water,
oil, glycerine -, the objective, the objective DIC prism - also known as
"auxiliary DIC prism" -, the DIC analyzer and finally the observation or
imaging optics.

What you describe as the "DIC prism behind the objective" most probably is
the auxiliary DIC prism, i.e. the DIC prism on the image side of the
objective. While a number of microscope producers have this auxiliary DIC
prism mounted so that you can adjust it for optimizing your contrast image
("DIC slider"), in the Olympus systems an adjustment is done in a slightly
different way; Olympus provides a lambda/4 plate as an additional element
between DIC polarizer and condenser DIC prism. The user then adjusts the
orientation of the DIC polarizer in order to change the contrast settings.
Also, optimal DIC observation sometimes may request a rotatable specimen
stage centered in the optical axis of the microscope.

The name DIC, "D"IC, "D"ifferential Interference Contrast, delivers the
explanation to your question. The DIC prism, also known as "Nomarski
prism", named after its inventor Jerzy Nomarski, provides a "d"ifferential
splitting of the ray paths of the light in the microscope. If you do
confocal laser scanning microscopy and have a well polarized laser beam -
normally, a laser beam generated by a "typical CLSM laser" will be
polarized per se, but it might nevertheless be sent through the DIC
analyzer (which is nothing else but a polarizer) -, then that beam will be
split into two beams, which are laterally displaced "differentially". The
lateral separation will hence be small, smaller than the lateral
resolution limit of the objective when used in the wide field observation
mode. Other types of interference contrast microscopes - e.g. Jamin
Lebedeff - will generate two beams with a macroscopic separation; then one
beam, the reference beam, will bypass the entire microscope on a ray path
outside the microscope frame, but this is not of any interest here.
What hence happens when you try to do CLSM with a fluorescent specimen -
fluorescent light assumed to be un-polarized - and have a DIC prism in the
ray path is that you will experience what are a kind of super imposed
"double images" if you have a sufficiently large zooming factor so that
you approach the resolution limit with your pixelization. The story is a
little bit more complicated in case of reflected light CLS microscopy
because of possible polarization conservation effects, but let's keep this
out of the discussion here for simplicity.

So, in order to avoid these effects, take out the secondary DIC prism from
the ray path when CLSMing.

NOW, there are some microscope objective holders, e.g. on a BX51WI used by
cell physiologists, e.g. Olympus item code WI-SRE2, in which a secondary
DIC prism, e.g. WI-DICTHRA2, is inserted so that you cannot easilry remove
it from the ray path. There does, however, exist a version of WI-SRE2,
named "FV10-SRE", if I recall correctly, where you have a slider, which
will permit you to easily insert or extract the DIC prism from the ray
path.

Unfortunately, this FV10-SRE seemingly is not sold a million times per
month or so, hence it is not really cheap. But it might help.

Best wishes,

Johannes



-- 
P. Johannes Helm, M.Sc. PhD
Seniorengineer
CMBN
University of Oslo
Institute of Basic Medical Science
Department of Anatomy
Postboks 1105 - Blindern
NO-0317 Oslo

Voice:	+47 228 51159
Fax:	+47 228 51499

WWW:	folk.uio.no/jhelm