Polarization Microscopy - Biomedical Optical Phase Microscopy and Nanoscopy - page 310

specimen area versus . We define contrast as the ratio between the intensity difference

between the specimen area and the background, divided by their sum:

θ

I spc 2 I bg
I spc 1 I bg

contrast 5

As apparent from Figure 15.5B , the highest contrast and therefore the best visibility of the
birefringent specimen are achieved when adjusting for a compensator angle of around θ min .

Figure 15.5B further shows the importance of using so-called high-extinction optics when
observing a specimen of low birefringence. Figure 15.5B introduces the parameter I min ,
which represents the spurious intensity observed when polarizer and analyzer are crossed.
The better the quality of the polarizers employed and of the intervening optical components
(especially condenser and objective lenses), the lower I min will be and the higher the
contrast that can be achieved for a specimen of given retardance. To improve the extinction
of microscope optics, it is possible to use the so-called polarization rectifiers, first
introduced by Inou´ and Hyde [9] , that counteract the polarization distortions introduced by
high numerical aperture (NA) objective and condenser lenses [22] . In the 1960s and 1970s,
some microscope manufacturers (e.g., American Optical Spencer and Nikon) made
rectified, high-extinction optics, with which high-contrast images of low-birefringent
objects, like the sperm head shown in Figure 15.1 , were recorded.

Since the 1980s, fluorescence microscopy has become the dominant technique for which the
optics of biological microscopes are optimized, demanding high transmission and low
image distortions over a wide range of wavelengths. These demanding specs are now met
by complex lens designs incorporating many components, each of which has special
antireflection coatings, in a single objective lens, compromising its polarization
performance. As a result, many modern objectives lead to low extinction and inferior
sensitivity when used in a polarizing microscope. While the LC-PolScope technique
discussed in Section 15.3 alleviates some of the shortcomings in polarization performance
of modern microscope objectives, it is still best to use microscope optics that is designated
for differential interference contrast (DIC) or polarized light microscopy.

15.3 The Liquid-Crystal Polarization Microscope (LC-PolScope)

Over the years, several schemes have been proposed to automate the measurement process
and exploit more fully the analytic power of the polarizing microscope. These schemes
invariably involve the traditional compensator, which is either moved under computer
control [23] or replaced by electro-optical modulators, such as Pockel cells [24] , Faraday
rotators [25] , and liquid crystal variable retarders [26] . These schemes also involve
quantitative intensity measurements using electronic light detectors, such as
photomultipliers or charge-coupled device (CCD) cameras. For quantitative measurements,