Page 156 - Fundamentals of Light Microscopy and Electronic Imaging

PRINCIPLES OF ACTION OF RETARDATION PLATES 139

cross is also useful in case a polar becomes misaligned in its holder, requiring the
operator to readjust the polar’s orientation.
• Using the telescope eyepiece or Bertrand lens, partially close the condenser aper-
ture diaphragm so that bright outer regions of depolarized light visible at the edge
of the aperture field are blocked. This greatly improves the extinction factor of the
microscope optics. Switching back to viewing mode, the field of view at extinction
should now be very dark. Normally it should only be necessary to rotate the speci-
men slide to examine the object at different azimuthal angles, leaving the positions
of the polarizer and analyzer fixed and unchanged. Because it is often necessary to
rotate specimens during examination, it is convenient to use a specially designed
rotating stage that is marked in degrees at its periphery. Rotating stages must be
adjusted so that their center of rotation is superimposed on the optic axis of the
microscope.

APPEARANCE OF BIREFRINGENT OBJECTS
IN POLARIZED LIGHT

Birefringent specimens exhibit characteristic patterns and orientations of light and dark
contrast features that vary, depending on the shape and geometry of the object (linear or
elongate vs. spherical) and the molecular orientation. In the absence of a compensator,
spherical objects with radially symmetric molecular structure exhibit a dark upright
polarization cross superimposed on a disk composed of four bright quadrants. Thus,
there are eight alternating bright and dark contrast regions distributed around the cir-
cumference of the sphere. If a compensator such as a /4 (quarter-wave) plate is inserted
into the beam so that its slow axis is oriented at 45° with respect to the transmission axes
of the polarizer and analyzer, a pattern of four quadrants is observed, with one pair of
opposite quadrants showing bright contrast and the other pair dark contrast. Instructions
for performing this operation are given at the end of the chapter.
Linear objects such as elongate striated muscle cells with coaxial alignments of lin-
ear filaments have a different appearance. In the absence of a compensator, rotation of
the specimen stage through 360° reveals eight angular azimuths at which the muscle
cells alternately appear bright (45°, 135°, 225°, and 315°) or dark (0°, 90°, 180°, 270°);
with a compensator present, there are four azimuths at 45°, 135°, 225°, and 315° at
which the object alternately appears light or dark with respect to the background.

PRINCIPLES OF ACTION OF RETARDATION PLATES
AND THREE POPULAR COMPENSATORS

With the addition of a retardation plate or compensator, the polarizing microscope
becomes an analytical instrument that can be used to determine the relative retardation
between the O and E waves introduced by a birefringent specimen. Since t (n n ),
e
o
either the birefringence or the thickness of a specimen can be determined if the other
parameter is known (see Chapter 8). An excellent description of the action of compen-
sators is given by Pluta (1993).
Transparent plates of birefringent materials such as quartz, mica, or plastic that
introduce a fixed amount of retardation between the O- and E-ray pairs are called retar-
dation plates or retarders. Retarders are prepared at a certain thickness and with the