Page 164 - Fundamentals of Light Microscopy and Electronic Imaging

PRINCIPLES OF ACTION OF RETARDATION PLATES 147

siderably less than the one wavelength retardation required to generate an interference
color. Thus, biological specimens such as living cells are frequently examined with the
green line of a mercury arc lamp with a /4 plate designated for this wavelength. The
use of monochromatic green light increases image contrast, allows more precise meas-
urements of retardation, and helps protect living cells.
If a birefringent specimen is positioned diagonally between two crossed polars,
the combined action of the specimen plus the λ/4 plate generates linearly polarized
waves whose E vectors are tilted at some azimuth depending on the amount of retar-
dation at the specimen. This action occurs because the λ/4 plate produces linearly
polarized light from incident, elliptically polarized waves. A rotation of the analyzer
through 180° is equivalent to a relative retardation by the specimen of one wavelength.
With every 90° of rotation of the analyzer, a birefringent object such as a fiber looks
alternately dark or bright. Since retardations of most cells and organelles are usually
less than 1 , and because the compensation method is so sensitive, Sénarmont com-
pensation is commonly used to analyze cellular specimens. Compensation with a /4
plate allows for measurement of relative retardations of up to 1 with an accuracy of
0.15 nm.
To measure , the /4 plate is inserted into the optical path between two crossed
polars as described. An elongated birefringent object (e.g., a muscle fiber containing
aligned myofibrils) is first oriented at a 45° angle with respect to the transmission axes
of the crossed polars with the rotating stage of the microscope. At this orientation, the
fiber generates elliptically polarized light, which is converted into linearly polarized
light by the /4 plate. These waves are partially transmitted by the compensator, caus-
ing the fiber to look bright against a maximally dark background. The analyzer is then
rotated from its crossed position through an angle until the fiber’s intensity appears
maximally dark (extinction) against a gray background. Notice that as the analyzer is
rotated to obtain extinction of the specimen, the background is brightening. Since the
angle of rotation of the analyzer at extinction is equal to one-half of the full phase shift
between the O and E rays, the relative retardation is given as

obj 2 .

can be used to calculate the values of the refractive index ellipsoid or the thickness of
the object if one of these two parameters is known, since t (n n ).
o
e
Brace-Koehler Compensator

For measuring very small phase retardations that occur in fine ultrastructural features
such as mitotic spindles, cleavage furrows, and stress fibers in living cells, a Brace-
Koehler compensator can be employed. This rotating compensator usually contains a
thin birefringent plate made of mica, whose optic axis is contained within the plane of
the plate. The compensator is used with monochromatic illumination—generally the
546 nm green line of the mercury arc lamp—and is capable of measuring retardations
up to the limit of the compensator (a fixed value, but ranging from /10 to /30 depend-
ing on the particular compensator) with an accuracy of 0.3 nm. These features make
the Brace-Koehler compensator a highly sensitive measuring device.
In this method of compensation the analyzer and polarizer remain fixed in position,
because the compensator itself is rotatable. The slow axis of the plate (marked on the