Polarization Microscopy - Biomedical Optical Phase Microscopy and Nanoscopy - page 312

compensator located between the light source (typically an arc lamp followed by an
interference filter) and the condenser lens. The analyzer for circularly polarized light is
placed after the objective lens. The universal compensator is built from two variable retarder
plates and a linear polarizer. The variable retarder plates are implemented as electro-optical
devices made from two liquid crystal plates. Each liquid crystal plate has a uniform
retardance that depends on the voltage applied to the device. An electronic control box that in
turn is connected to a computer supplies the voltage. The computer is also connected to the
electronic camera, typically a CCD camera, for recording the specimen images projected
onto the camera by the microscope optics. Specialized software synchronizes the image
acquisition process with the liquid crystal settings and implements image-processing
algorithms. These algorithms compute images that represent the retardance and slow axis
orientation at each resolved image point. For a more in-depth discussion of the LC-PolScope
technology, see Refs. [18,19,26] . Shribak also proposed a variance to the LC-PolScope
technique that uses only one liquid crystal device instead of two [27] .
The commercial LC-PolScope technique, developed and distributed by CRi Inc. (now part of
Caliper Life Sciences and PerkinElmer), is available as an accessory to microscope stands of all
the major microscope manufacturers (e.g., Leica, Nikon, Olympus, and Zeiss). It usually
includes the universal compensator, circular polarizer, a camera with control electronics, and a
computer with software for image acquisition and processing. Three slightly differing versions
are available, each optimized for research in the life sciences (Abrio LS), industrial metrology
(Abrio IM), and for in vitro fertilization and related laboratory techniques (Oosight).
Figure 15.7 shows retardance images recorded with the LC-PolScope, illustrating the
clarity, resolution, and analytic potential for examining the birefringent fine structure in
living cells. Figure 15.8 shows its use in procedures involving enucleation and cloning.
All images were recorded without the need for exogenous stain or label in the specimen.
15.4 Practical Considerations
15.4.1 Choice of Optics
As indicated earlier, the polarization distortions introduced by the objective and condenser
lenses limit the extinction that can be achieved in a polarizing microscope. Most
microscope manufacturers offer lenses that are designated “Pol” to indicate low polarization
distortions, which can arise from a number of factors, including stress or crystalline
inclusions in the lens glass and the type of antireflection coatings used on lens surfaces.
Some lens types are available only with “DIC” designation. “DIC” lenses do not meet the
more stringent “Pol” requirements but pass for use in DIC and can also be used with the
LC-PolScope (without the Wollaston or Nomarski prisms specific to DIC, of course).