Page 165 - Fundamentals of Light Microscopy and Electronic Imaging

148 POLARIZATION MICROSCOPY

compensator mounting) is oriented parallel to the north-south transmission axis of the
analyzer and corresponds to the 0° position on the compensator dial. A linear birefrin-
gent specimen, such as a bundle of actin filaments or microtubules, is first oriented at a
45° angle with respect to the transmission axis of the analyzer by rotating the stage of
the microscope to give maximum brightness. Because the maximum optical path differ-
ence of the compensator is small ( /20), the background appears dark and remains rel-
atively dark through different angles of rotation of the compensator. The compensator is
then rotated counterclockwise from its 0° position until light from the specimen is
extinguished, matching the dark background. The angle of rotation from the zero posi-
tion on the compensator is used to calculate the relative retardation between the O and
E rays using the equation

obj comp sin 2 ,

where is the angle of rotation of the compensator and comp is the maximum optical
path difference of the compensator ( /20). The precise value of comp must be deter-
mined by calibration and is a constant in the equation. Depending on the particular com-
pensator, retardations of /2000 can be measured under optimal conditions.

Exercise: Determination of Molecular Organization in
Biological Structures Using a Full Wave Plate Compensator

First prepare the microscope for polarization microscopy. The analyzer and polar-
izer might already be installed in the microscope, in which case it is only neces-
sary to bring them into position as described in the text. If not, strips of dichroic
polarizing film can be used in the vicinity of the specimen slide, although this is
not standard practice for a research grade instrument. The polarizer is mounted
between the specimen slide and the condenser lens with its transmission axis ori-
ented east-west. The analyzer is placed between the specimen slide and the objec-
tive lens with its transmission axis oriented north-south. The analyzer is rotated
slightly until the background is maximally dark (extinction). If a red-I plate is
used, it is inserted between the crossed polars with its slow and fast axes oriented
at a 45° angle with respect to the transmission axes of the polars. (See Appendices
II and III for sources and instructions on how to prepare red-I plates for this exer-
cise.) The blackened edge of the red-I plate marks the direction of the slow axis
of the wavefront ellipsoid.
For orientation, examine several birefringent materials between crossed
polars without the red plate, including grains of corn starch, plant cell crystal-
loids, insect leg and flight muscle, and prepared slides of striated muscle, butter-
cup root, and pine wood. Notice that linear birefringent structures such as
myofibrils in striated muscle and plant cell walls are brightest when their long
axes are oriented at 45° with respect to the transmission axes of the crossed
polars. Spherical particles like starch grains are bright and exhibit a dark upright
extinction cross through their centers. Re-examine the specimens after inserting
the red plate. If the waveplate is a red-I plate, the background has a bright
magenta-red (first order red) interference color. (Note: Other interference colors