Polarization Microscopy - Biomedical Optical Phase Microscopy and Nanoscopy - page 308

retardation, also called retardance, and thereby quantify the magnitude and orientation of
molecular order in the specimen. Retardance is an extrinsic property of the material and a
product of the birefringence and the path length l through the material:

retardance 5 R 5 Δn U l

Birefringence also has an orientation associated with it. The orientation refers to the
specimen's optical axes of which one is called the fast axis and the other the slow axis .
The light polarized parallel to the slow axis experiences a higher refractive index and travels
slower than the light polarized parallel to the fast axis. In materials that are built from aligned
filamentous molecules, the slow axis is typically parallel to the average orientation of the
filaments. Birefringence orientation always correlates with molecular orientation, which
usually changes from point to point in the specimen (see aster in Figure 15.4 ).

15.2.3 Quantitative Analysis of Specimen Retardance

Birefringence and retardance are optical properties that can be directly related to molecular
order such as the alignment of polymeric material. By using the traditional polarizing
microscope, Sato, Ellis, and Inou´ measured the retardance of mitotic spindles in living
cells and were able to conclude that the birefringence is caused by the array of aligned
spindle microtubules [3] . Their measurements were made possible by the careful analysis of
specimen birefringence using a traditional compensator in addition to a polarizer and
analyzer in the microscope optical path. Using a Brace-K¨hler compensator, the retardance
of a resolved image point or uniformly birefringent area in the field of view can be
measured by carrying out the following steps:

1. With the specimen in place and by viewing through the eye piece (or on the monitor
attached to a video camera), make sure that polarizer and analyzer are crossed and the
compensator is in the extinction position. Given those settings, background areas that
have no birefringence appear dark, and birefringent structures change from dark to
bright four times when rotating the specimen by 360 .
2. Again, using the rotatable stage, orient the birefringent structure of interest so that it
appears darkest (extinction position), and note the orientation and then rotate the
specimen 45 away from the extinction position. At that orientation, the structure appears
brightest. (By eye, it is usually easier to determine the orientation that leads to the lowest
intensity rather than the highest intensity. The two orientations are 45 apart.)
3. Now rotate the compensator either clockwise or counterclockwise, until the birefringent
structure of interest appears dark. When rotating the compensator, any background
area becomes brighter, while birefringent structures become either brighter or darker.
For structures that turn darker than the background, the birefringence of the
compensator is said to “compensate” the birefringence of the sample structure. Take the