Page 166 - Fundamentals of Light Microscopy and Electronic Imaging

PRINCIPLES OF ACTION OF RETARDATION PLATES 149

are possible if the thickness of the plate is not carefully controlled; in this case the
interference colors of objects will vary somewhat from the colors described in the
exercise.) Muscle fibers, collagen bundles and elongate plant crystals look purple
(an addition color) if their long axes are parallel to the slow axis of the wavefront
ellipsoid of the red-I plate, and golden yellow (a subtraction color) if their long
axes are perpendicular to the slow axis of the plate. Looking at the Michel Lèvy
chart of polarization colors, it can be seen that the relative retardation between the
purple and gold colors is about 100 nm. Further explanation of the interference
colors is given below and in the text.
We will now use the red-I plate to determine the molecular orientation in two
plant cell inclusion bodies. Amylose-containing starch storage granules and
lignin-containing wood pits are birefringent spherical objects in plant cells con-
sisting of parallel bundles of long-chain molecules and polymers. The two likely
patterns for molecular order in these structures might be compared, respectively,
to the needles projecting radially from a pincushion (radial pattern) or to surface
fibers in the layers of an onion (tangential pattern). The axis of carbon-carbon
bonds in these models differs by 90° and is easily observed in a polarizing micro-
scope equipped with a plate even though the inclusion bodies are extremely
minute. The microscope is set up with the slow and fast axes of the wavefront
ellipse of the plate oriented at 45° with respect to the transmission axes of the
crossed polars. The specimen background exhibits a bright magenta-red color,
whereas the granular inclusion bodies appear as spheroids with opposite pairs of
yellow-orange and purple-blue quadrants. It is remarkable that the yellow and
blue color patterns are reversed for the two types of bodies, indicating differences
in the pattern of molecular alignment! Each pair of yellow quadrants and blue
quadrants depends on the orientation of the slow and fast axes of the wavefront
ellipsoid of the plate. The blue quadrants (the addition color) indicate the
azimuth along which the slow axes of the specimen and plate are parallel to one
another; the yellow quadrants (the subtraction color) indicate the azimuth along
which the slow axes of the plate and object are perpendicular. By constructing a
diagram where molecular alignment in each quadrant is shown as a series of par-
allel lines, you can deduce whether the molecules project radially like a pincush-
ion or are ordered tangentially like the layers of an onion.
Roots of herbaceous plants contain an epidermis, a cortex, a pericycle (pro-
liferative tissue), and a vascular cylinder or stele that runs along the axis of the
root (Fig. 9-7). Inside the vascular cylinder, identify the xylem—long, longitudi-
nally oriented elements for conducting water and dissolved nutrients and miner-
als principally upward to the leaves and branches. The phloem transports
macromolecules and metabolites (principally downward toward the roots). These
are surrounded by a sheath of pericycle and endodermis cells. Outside the peri-
cycle is an extensive layer of cortex containing starch storage granules. Notice the
specific stains for the xylem and phloem cells. The walls of plant cells contain
ordered filaments of cellulose and lignin and thus are highly birefringent in a
polarizing microscope.
The section of pine wood contains mostly xylem (the water transport tissue),
plus some vascular rays and pitch canals where pitch accumulates (Fig. 9-7). The