Polarization Microscopy - Biomedical Optical Phase Microscopy and Nanoscopy - page 316

index as a function of the polarization state of the illuminating light, DIC imaging highlights
refractive index changes as a function of position in the specimen. Since DIC employs
polarized light, its image contrast tends to combine both effects, i.e., image features in DIC
reflect both types of changes in the specimen refractive index. Shribak has developed
powerful new DIC methods, described in Chapter 2, which includes a variance that combines
DIC with polarized light imaging [29] . Nevertheless, many microscopes are equipped with
traditional polarization and DIC optics, or LC-PolScope and DIC optics, and the operator
might wonder how to combine both types of observations and get optimal results. Therefore,
I briefly comment on their combination.
For DIC imaging, two matching prisms are added to the polarization optical train. The two
prisms, called Wollaston or Nomarski prisms, are specially designed to fit in specific
positions, one before the condenser and the other after the objective lens, and to match
specific lens combinations. The prisms in their regular positions are usually combined with
the linear polarizers of a traditional polarizing microscope. The prisms can also be combined
with the LC-PolScope setup, in which case, the universal compensator and circular analyzer
take the places of the linear polarizer and analyzer in a standard DIC arrangement. In fact, the
same DIC prisms function equally well using either linearly or circularly polarized light.
Note, however, that Carl Zeiss has recently combined linear polarizers with their condenser
DIC prisms preventing their use with circularly polarized light.
In combining DIC with the LC-PolScope, the universal compensator is first calibrated
without prisms to find the extinction setting. With the compensator set to extinction, the
prisms are entered into the optical path. Typically, one of the two prisms has a mechanism
for finely adjusting its position, which in turn affects the brightness and contrast of the DIC
image. In a PolScope setup, the mechanical fine adjustment or the universal compensator
can be used to add a bias retardance to change the brightness and contrast of the DIC
image. When using the universal compensator, retardance must be added or subtracted from
the extinction setting of either the LC-A or LC-B retarder ( Figure 15.6 ), depending on the
orientation of the shear direction of the prisms.
For live cell imaging, it is very useful to be able to switch easily between polarized light
and DIC imaging. DIC provides good contrast of many morphological features in cells
using direct viewing through the eyepiece or by video imaging. When viewing specimens
that have low polarization contrast, it is more effective to align the optics, including the
visualization of the specimen, using first DIC. After the optics has been aligned and the
specimen is in focus, the DIC prisms are removed from the optical path and the PolScope
specific adjustments are completed.
Fluorescence imaging can be combined with the PolScope in several ways. Fluorescence is
commonly observed using epi-illumination, which requires a filter cube in the imaging path.
The filter cube includes a dichromatic mirror and interference filters for separating the