Polarization Microscopy - Biomedical Optical Phase Microscopy and Nanoscopy - page 303

microscope [2] . These figures are generated by the birefringence of parallel arrays of MTs
that form the mitotic spindle and astral rays in dividing eukaryotic cells [3] . In addition to
the mitotic spindle, the images in Figure 15.2 also show the strong birefringence of the
fertilization envelope that surrounds the cell and is formed by tangentially aligned filaments
and paracrystalline polymer layers [4] .

The birefringent structures seen in Figures 15.1 and 15.2 exhibit the orientation-dependent
contrast that is a hallmark of the traditional polarizing microscope. Figure 15.3 shows an
image recorded with a new type of polarized light microscope, the liquid-crystal polarization
microscope (LC-PolScope), which avoids the confusion of orientation-dependent contrast.
The LC-PolScope generates a map that represents the magnitude of birefringence and
separately a map of its orientation. Figure 15.3 shows only the magnitude of birefringence, or
retardance (see Section 15.7 ), independent of its orientation in the specimen. Black in a
retardance image means that there is no anisotropy at that image point, and increasing
brightness means increasing retardance, using a linear relationship between image brightness
and the measured retardance values. This is in stark contrast to traditional polarized light
micrographs, in which image brightness depends on both the magnitude of birefringence and
its orientation with respect to the polarizers and compensator in the optical path. Using a
traditional polarizing microscope one can interpret the specimen anisotropy only through a
series of observations while stepwise rotating the specimen and/or the polarizers and
compensator. The liquid crystal-based universal compensator in the LC-PolScope relieves the
experimenter from manually rotating optical components and at the same time combines
several polarization optical views of the specimen into one resultant image that represents the
measured retardance and/or orientation of the specimen birefringence. Using this new
technique, very small retardance values (down to 0.03 nm) can reliably be measured, in fast
time intervals (less than 1 s), over the whole field of view at the highest resolution of the light
microscope (200 nm). The retardance image of the dividing insect spermatocyte in
Figure 15.3 illustrates the sensitivity and analytic power of the LC-PolScope that provides
comprehensive measurements of intrinsic material properties of native anisotropic structures
inside a living cell. In fact, the structural origins of the birefringence of many of the
organelles visible in Figure 15.3 , such as mitochondria and lipid droplets, remain mysterious.

The classic topics on polarizing microscopy were published several decades ago, when
optical phase microscopy was in its heyday [5,6] . Several decades before Hartshorne,
Schmidt published two celebrated monographs that to this day report the most
comprehensive surveys of biological specimens observed under polarized light [7,8] .
Inspired by Schmidt's observations, Shinya Inou´ significantly improved the sensitivity of
the instrument and made seminal contributions to our understanding of the architectural
dynamics inside living cells [9

11] . For many lucid discussions of polarized light
microscopy and its application to biology, I refer the reader to the many articles and topics
by Inou´, including his recently published Collected Works [12 16] . Finally, I refer to my