Differential Interference Contrast (DIC)

Polarized light passing through this prism is sheared into two closely spaced waves with different angles of deflection, the ordinary and the extraordinary wave. They have perpendicular planes of polarization and for these two waves the medium behaves as if it has a single effective refractive index. The shear of the wavefronts takes place at the interference plane that lies at the refractive index junction between the two prisms. The light waves are spatially separated by the shear angle. Their direction and the distance between them are the same for all matching light waves across the whole prism. The space between these two waves has to be smaller than the resolution limit of the microscope.

So the specimen is illuminated by closely spaced pairs of light waves that enter parallel with a lateral displacement. If these two light waves interact with specimen parts with different refractive indices or different thickness, there will be a difference in their optical path length as they emerge from the specimen. The optical path length is the product of the refractive index and the thickness between two points on an optical path. It is related to the transit time and the velocity of light. As the optical path length is related to the transit time of the light, a phase shift occurs between the two matching light waves.

After the light waves have traversed the specimen they are brought back together via another Wollaston prism. Now they interfere with each other, forming elliptically polarized light. The elliptically polarized light passes through an analyzer. Only light with a distinct polarization plane is able to pass and therefore different amplitudes are produced for the different light waves. The phase shift is transformed into an amplitude shift which leads to different light intensities in the resulting image.

For modern DIC microscopes the Wollaston prism is often modified. One example is the Nomarski prism. It consists of two birefringent wedges as well but only one wedge is identical to the one in a Wollaston prism. The other one is modified and the optical axis is obliquely positioned. This leads to an interference plane which lies outside the prism. So the prism can be located outside the objective’s aperture plane and is easier to use.

In DIC microscopy the phase shift differences of neighboring object points contribute to the image formation. Details of the specimen which have a gradient in their refracting indices or their thickness are visualized. As the light beams are polarized perpendicularly to each other different images can be produced by rotating the microscope stage.

It is important to remember not to use plastic in DIC microscopy because many polymers have a depolarizing effect on light and would destroy the contrast.

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