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Light Microscopy: Comparison of Optics

Author(s): David R. Caprette, PhD

Differential Interference Contrast

Differential interference contrast (D.I.C.) optics also are known as Nomarski optics, after their inventor, Jerzy Nomarski. As with phase contrast microscopy, D.I.C. enhances contrast by taking advantage of differences in refractive index among parts of a specimen. In D.I.C., however, the optics make use of polarized light.
Light consists of electromagnetic waves that have the properties of particles as well as of waves. Suppose you view a beam of white light coming directly toward you from the source. If you could see the individual waves somehow, you would see them vibrating in random directions. If, on the way to you, the beam passed through a polarizing filter, the waves that pass through would be seen to vibrate only in one plane, that is, in two opposite directions such as up and down, left and right, or at some other angle, depending on the position of the filter. Without going into too much detail, let's just acknowledge that by using polarizing filters, one can direct more than one beam of light at an object and select one or the other beam using appropriate filters.
D.I.C. works by using a prism to direct two beams of polarized light at a specimen, with one beam slightly offset from the other. A second prism reassembles the beams to produce a shadowing effect. The result is an apparent three-dimensional image, not unlike a scanning electron micrograph or the three-dimensional effect one sees when viewing the terminator on the Moon. The terminator is the boundary between the lit and unlit face of the Moon, where the shadowing effect highlights craters and other features. In D.I.C., the contrast we see is due to differences in refractive index within a specimen, rather than to topography. In fact, the three-dimensional image does not necessarily correspond to the actual shape of the specimen.
A D.I.C. microscope typically is very expensive and not likely to be used in a teaching laboratory. However, one can mimic a D.I.C. effect using a bright field microscope with an adjustable condenser. It is necessary to move the condenser slightly off axis to produce a shadowing effect. A very dramatic effect can be achieved through trial and error.