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3.4  Nonfluorescence Microscopy

retardation that occurs in each polarization direction and thus generates information about

the relative orientation of spatially periodic molecular structures of the birefringent sample.

Some of these structures are involved in mechanical features of tissues and cells, and thus

polarization microscopy can be used to probe biomechanics (see Chapter 6).

Differential interference contrast (DIC) microscopy is a related technique to polarization

microscopy, in using a similar interference method utilizing polarized light illumination on

the sample. A polarizer again is placed between the VIS light source and condenser lens,

which is set at 45° relative to the optical axis of an additional birefringent optical compo­

nent of either a Wollaston prism or a Nomarski compound prism, which is positioned in the

beam path in the front focal plane of the condenser lens. These prisms both generate two

transmitted orthogonally polarized rays of light, referred to as the “ordinary ray” (polarized

parallel to the prism optical axis) and the “extraordinary ray,” thus with polarization E-​field

vectors at 0°/​90° relative to the prism optic axis. These two rays emerge at different angles

relative to the incident light (which is thus said to be sheared), their relative angular separ­

ation called the “shear angle,” due to their different respective speeds of propagation through

the prism (Figure 3.3a).

These sheared rays are used to form a separate sample ray and reference ray, which are both

then transmitted through the sample. After passing through the sample and objective lens,

both transmitted sheared rays are recombined by a second matched Wollaston/​Nomarski

prism positioned in a conjugate image plane to the first, with the recombined beam then

transmitted through a second polarizer analyzer oriented to transmit light polarized at 135°.

FIGURE 3.3  Generating image contrast in light microscopy. (a) Schematic of differential interference contrast illumin­

ation. (b) Typical fluorescence microscopy filter set. (c) Wavelength selection using a Fabry–​Perot interferometer design.

(d) Absorption and emission spectra for GFP, with overlaid excitation and emission filters and dichroic mirror in GFP filter set.