Page 175 - Fundamentals of Light Microscopy and Electronic Imaging

158 DIC MICROSCOPY AND MODULATION CONTRAST MICROSCOPY

Nomarski cemented a conventional wedge with its optic axis parallel to the surface
of the prism to another specially cut wedge whose optic axis was oriented obliquely with
respect to the outside surface of the prism. For the condenser lens, there are fewer spatial
constraints, and a conventional Wollaston prism can sometimes be used. In many cases
the condenser prism is a modified Wollaston prism as well, although its interference
plane is usually closer to the prism. Thus, the two Wollaston prisms of the DIC micro-
scope are cut differently and are not interchangeable. The condenser prism acts as a beam
splitter, while the objective prism recombines the beams and regulates the amount of
retardation between O and E wavefronts. Note the requirement for Koehler illumination
to correctly position the interference planes of the DIC prisms in the conjugate aperture
planes of the condenser and objective lenses. The central or 0th-order interference fringe
seen in the interference pattern just described is used to determine the proper orientation
of the prisms during microscope alignment and is discussed later in this chapter.

Demonstration:The Action of a Wollaston Prism
in Polarized Light

• Place a DIC prism between two crossed polars on a light box (white light)
and rotate the prism until a dark interference fringe is observed. The intense
dark band seen in the middle of the prism is the central 0th-order fringe that
results from destructive interference between the O and E wavefronts that
have equal path lengths in the middle of the prism. Prisms intended for low-
magnification, low-NA optics reveal several parallel interference fringes.
Prisms intended for high-magnification, high-NA work reveal a single dark
interference fringe.
• Observe that the higher-order fringes appear in the colors of the interference
spectrum if white light is used. Higher-order fringes appear dark gray instead
of black when illuminated with monochromatic light.
• Also notice that the interference fringes appear to float in space some millime-
ters above or below the prism. This is because modified Wollaston (Nomarski)
prisms have their interference planes located outside the prism itself.
• Rotate the prism between the crossed polars again and notice that there is a
unique position giving extinction. At this orientation the phase retardation
introduced between O and E rays by one slab of the prism is exactly reversed
by phase displacements in the other slab, so light emerges plane parallel and
vibrating in the same plane as the incident polarized light. The resultant rays
are blocked by the analyzer, giving extinction.
• In the microscope examine the action of a DIC prism between crossed polars
(the other prism has been removed) in the aperture plane using a telescope eye-
piece or Bertrand lens. A bright field is seen with a dark fringe running across
it. When both prisms are in position and the back aperture is observed, the field
looks dark (extinction) because light emerging from each position in the con-
denser prism is now exactly compensated for by the objective prism. All beams
emerging from the objective prism are again linearly polarized in the direction
of the original polarizer, and thus are blocked by the analyzer (extinction).