Page 160 - Fundamentals of Light Microscopy and Electronic Imaging

PRINCIPLES OF ACTION OF RETARDATION PLATES 143

the spectral colors are seen except for a band of wavelengths in the green portion of the
spectrum. If now a birefringent object such as a sheet of cellophane is inserted together
with the red-I plate between two crossed polars and rotated, the color shifts to blue and
yellow. This is because the relative retardation for the combination of red plate plus
object is now greater or less than 555 nm, causing the wavelength of linearly polarized
light to be shifted and resulting in a different interference color. By comparing object
colors with the background color of the plate and referring to a Michel Lèvy color chart
(Color Plate 9-1), it is possible to determine the relative retardation introduced by the
object.
For birefringent biological materials such as small spherical inclusion bodies, full-
wave plates can be used to determine patterns of molecular arrangement (see exercise on
plant cell inclusion bodies). The shifted blue and yellow colors are called, respectively,
addition and subtraction colors, and immediately tell about the orientation and magni-
tude of the fast and slow axes of the specimen’s index ellipsoid. Thus, the plate is a
useful semiquantitative device for estimating minute retardations and the orientations of
index ellipsoids. As a survey device used to examine complex specimens, it may be
more convenient than other more precise compensators.

Demonstration: Making a Plate from a Piece of Cellophane

Crumple up a piece of cellophane and place it between two crossed polars on a
light box. It is immediately obvious that cellophane is strongly birefringent. In
regions where the sheet is a single thickness, the color of the birefringence is
white, but in regions where the sheet is folded over on itself and overlapping, a
surprising display of interference colors is observed. The colors indicate regions
where the phase retardation between O and E waves subtends a whole wavelength
of visible light, resulting in the removal of a particular wavelength and the gener-
ation of a bright interference color. In these colorful regions, cellophane is acting
as a full-wave plate. The different colors represent areas where the amount of rel-
ative retardation varies between the O and E waves. The optical path correspon-
ding to any of these colors can be determined using a color reference chart (Color
Plate 9-1). If we now insert an unknown birefringent object in the path, the color
will change, and using the chart and the cellophane wave plate as a retarder, we
can examine the color change and make a fairly accurate estimate of the path
length of the unknown specimen. Used this way, the cellophane wave plate acts
like a compensator. The following demonstration shows how to make a cello-
phane wave-plate retarder, understand its action, and use it as a compensator:

• Place a sheet of clear unmarked cellophane between two crossed polars on a
light box and rotate the cellophane through 360°. Use a thickness of the type
used for wrapping CD cases, boxes of microscope slides, pastry cartons, and
so forth. Cellophane dialysis membranes are also very good. Do not use the
plastic wrap made for food products or thick, stiff sheets. There are four
azimuthal angles (located at 45° with respect to the crossed polars) at which
the cellophane appears bright against the dark background. Mark the loca-
tions of these axes by drawing a right-angled X on the sheet with a marking