Page 162 - Fundamentals of Light Microscopy and Electronic Imaging

PRINCIPLES OF ACTION OF RETARDATION PLATES 145

since this is manufactured with the strands of cellulose (and direction of the
slow axis of the wavefront ellipsoid) parallel to the length of the tape.
Remove two 1 cm length pieces of tape and draw a wavefront ellipse on each
piece. The slow (short) axis of the ellipse is parallel to the long axis of the
strip of tape; the fast (long) axis is parallel to the cut edge of the tape. Place
the two pieces of tape at 90° to each other on the folded piece of cellophane,
with each piece of tape being parallel to the edge of the cellophane sheet.
Place the folded sheet between the crossed polars and orient the sheet at a
45° angle. The regions covered with tape show two new interference col-
ors—pale yellow and sky blue—superimposed on a bright yellow back-
ground. Reference to the polarization color chart shows that the blue color
corresponds to the removal of a wavelength of 690 nm (460 230 nm). This
can only occur if the retardation caused by the folded cellophane is further
retarded by the tape. The slow axes of the tape and cellophane must be par-
allel to each other. Conversely, the pale yellow interference color corre-
sponds to removal of a much shorter wavelength (460 230 230 nm). The
slow axis of the tape must be perpendicular to the slow axis of the folded cel-
lophane sheet. This causes the net retardation to be reduced, thus producing
a pale yellow interference color. The ellipse drawn for the tape that appears
blue can now be marked, retaining the same orientation, on the original cel-
lulose sheet. We now have a calibrated cellulose yellow-I retarder that can be
used to determine the amount of retardation and the orientation of the slow
and fast axes of other birefringent materials.
• Further practice with the yellow-I plate will reinforce the concept of the
compensator as a device for increasing or diminishing the relative phase
retardation between the O and E rays and measuring the relative retardation
of unknown birefringent objects.

Sénarmont Compensator

Compensation by the method of de Sénarmont requires a fixed quarter-wave plate ( /4
plate) and a rotatable analyzer. Since /4 plates are designed for use at a specific wave-
length, microscope illumination must be monochromatic, typically at the 546 nm green
line of a mercury lamp, although /4 plates intended for use with other wavelengths,
such as the 589 nm yellow line of the mercury lamp, are also available. Sénarmont
compensation is commonly used to measure the amount of retardation in biological
specimens, such as cell organelles, plant cell walls, and muscle fibers, that induce
retardations between /20 and 1 . As explained, this compensator is also used to
improve the visibility of birefringent specimens, because birefringent objects can be
made to produce bright and dark contrast patterns against a medium gray background
(Fig. 9-1).
The retarder ( /4 plate) is mounted in a fixed orientation between two crossed
polars, usually between the back aperture of the objective lens and the analyzer (Fig. 9-6).
The analyzer is rotatable and is marked in degrees around its circumference so that the
angle of rotation can be precisely determined. The /4 plate is made of a birefringent
material such as quartz and is prepared so that the optic axis of the plate is parallel to the