Differential Interference Contrast microscopy "DIC"

Like Phase Contrast, DIC translates sample Optical Path Length into contrast that can be visualized. In DIC contrast is generated when two "difference images" are recombined within the microscope. The two images are created when image rays traverse the birefringent Wollaston Prism I. The two wavefronts (e- and o-ray) will be separated spatially (shear, d) by a small amount (less than resolvable) but in phase with each other (due to the geometry of the prism). When these two fronts pass through the sample, two slightly phased "difference images" are formed. These images are subsequently recombined by (the inverted) WP2. This second prism introduces additional phasing "bias", or phase differential between the two images. However, they are vibrating at orthogonal planes and do not yet interfere. Superposition of the difference images by the Analyzer allows wave interference to increase or decrease intensity, thus generating contrast.

The image looks 3D, due to a shading appearance caused by the overlapping, but "sheared" difference images. This results in constructive interference on one side and destructive on the other. Translating (moving sideways) WP2 changes relative phase of the two wavefronts, which results in contrast changes in the interference image

DIC is a very good optical technique for looking at thin, clear objects. The resolution is higher than is possible with Phase Contrast, and with no Phase Halo artifact.

DIC is based on the concept of an "Interferometer" whereby a beam of light is split into two beams. One passes to the optical image plane unmodified, whereas the other interacts with an unknown sample and is phase-shifted. Subsequent recombination of the "Reference" and "Sample" beams allows wave interference to occur, resulting in light or dark regions (corresponding to constructive and destructive interference). A modification of this is to place the sample in front of the beam splitter and (slightly) offset the reflecting mirrors. When the "tilted" difference images interact at the image plane, gradients of contrast are generated (b)
A DIC microscope consists of two, inverted, Wollaston (or Nomarski) prisms, plus a Polarizer and Analyzer. Contrast is generated by the interaction of two closely separated, but non-overlapping images of the sample.