Polarization Microscopy - Biomedical Optical Phase Microscopy and Nanoscopy - page 307

Compensator

While not absolutely necessary for some basic observations, especially of highly birefringent
objects, the compensator (a) can significantly improve the detection and visibility of weakly
birefringent objects, (b) is required to determine the slow and fast axis of specimen
birefringence, and (c) is an indispensable tool for the quantitative measurement of object
birefringence. There are several types of compensators; most of them are named after their
original inventors. For the observation of weakly birefringent objects, typically encountered
in biological specimens, the Brace-K¨hler compensator is most often used. It consists of a
thin birefringent plate, often made from mica, with a retardance of a tenth to a thirtieth of a
wavelength ( /30; see Section 15.7 for the definition of retardance). The birefringent

plate is placed in a graduated rotatable mount and inserted either between the polarizer and
condenser, as in Figure 15.4 , or between the objective lens and the analyzer. The location
varies between microscope manufacturers and specific microscope types. In either location,
the effect of the Brace-K¨hler compensator on the observed image is the same and its
standard usage is independent of its location. In general, the birefringence of the compensator
causes the image background to become brighter, while birefringent specimen parts can turn
either brighter or darker than the background, depending on their orientation with respect to
the compensator. If the birefringent structure turns brighter, its slow axis aligns more parallel
to the compensator slow axis, while if the structure turns darker, its slow axis aligns more
perpendicular to the compensator slow axis. Hence, the compensator with known slow axis
orientation can be used to determine the slow axis of birefringent parts in the specimen.
The compensator can also enhance specimen contrast, and it is used to quantify specimen
birefringence by measuring its retardance, as discussed in Section 15.2.2 .

λ /10 λ

15.2.2 Birefringence, Retardance, and Slow Axis

Birefringence occurs when there is a molecular order, when the average molecular
orientation is nonrandom, as in crystals or in aligned polymeric materials. Molecular order
usually gives the material two orthogonal optical axes, with the index of refraction along
one axis being different from the other axis. The difference between the two indices of
refraction is called birefringence and is an intrinsic property of the material:

birefringence 5 Δn 5 n O 2 n \
where n jj and n \ are the refractive indices for the light polarized parallel or perpendicular
to the two optical axes.

The light polarized parallel to one axis travels at a different speed through the sample than
the light polarized parallel to the orthogonal axis. As a result, these two light components,
which were in phase when they entered the sample, are retarded at a different rate and exit
the sample out of phase. Using the polarizing microscope, one can measure this differential