When a differential interference contrast (DIC) optical system is illuminated with white light from a tungsten-halogen lamp or similar source, the background (as observed through the eyepieces) can be varied from black through various shades of gray to higher order interference colors, even when no specimen is present. This interactive tutorial explores how changes to the optical path difference between orthogonal wavefronts, induced by translating the objective Nomarski prism, can produce a wide spectrum of interference colors that are useful in determining path lengths and for the optical staining of transparent specimens.

The tutorial initializes with two coherent wave packets of white light, which are represented by gray sine waves (initially the sine waves are overlapped, so only a single wave appears in the window). One wave is light gray in color while the other wave is dark gray. In order to reduce the complexity of the tutorial (and to clarify explanation of the concept) only three component wavelengths of white light are employed to represent the entire spectrum: Blue (400 nanometers), Green (550 nanometers), and Red (700 nanometers). Each colored wave in the tutorial (blue, green, and red) represents the resultant wave that occurs from either constructive or destructive interference of the coherent gray waves. A reference point on the abscissa (the Phase axis) denoted by S(1) represents the relative wavefront phase position when no optical path difference is applied to the system.

In order to operate the tutorial, use the mouse cursor to shift the Optical Path Difference (OPD or D) slider to the right from its default far left position. Changing the value of this slider mimics the effects of translating the objective Nomarski prism across the optical path in order to generate bias retardation in a DIC microscope. As the slider is shifted to the right, the induced optical path difference is presented above the slider (in nanometers) and also in the tutorial window as an expanded path length between the reference wavefront (S(1)) and the retarded (or advanced) wavefront (S(2)), which occurs when bias retardation is added to the optical system. In addition, Michel-Lévy colors produced by interference of the recombined wavefronts at the microscope analyzer are presented in the lower right-hand side of the tutorial window (the Interference Color box). The range of the Optical Path Difference slider is zero (no bias retardation) to 600 nanometers.

At small optical path differences (less than 140 nanometers) the amplitude of the red, green, and blue wavefronts is larger than the individual gray wave packets, a phenomenon that results from constructive interference. Between 140 and 275 nanometers, destructive interference effects take over and the amplitude of recombined waves is decreased. When the optical path difference is equal to a whole or multiple of a single white light component wavelength (in the tutorial, either 400, 550, or 700 nanometers), then both the light and dark gray sine waves have the same phase and constructive interference doubles the amplitude for that wavelength. However, at values equaling half of a component wavelength (200, 275, and 350 nanometers), the two wave packets (gray and light gray sine waves) are 180 degrees out of phase, resulting in complete destructive interference. Thus, at 200 nanometers, for example, white light is deprived of the blue component (having a wavelength of 400 nanometers). The same results occur for the green and red components at 275 and 350 nanometers, respectively. When one of the primary components is missing from white light, the remaining wavelengths are mixed uniformly according to their amplitude ratios to yield a unique interference color.

Because the amplitude ratio of the white light components varies with optical path difference, each path difference is associated with a characteristic interference color, which can be determined by examining a Michel-Lévy color chart. As discussed above, translating the objective Nomarski prism across the microscope optical axis produces a constant optical path difference between wavefronts traveling through the optical system from the condenser prism to the objective prism. Introduction of this optical path difference results in the generation of interference colors when vector components from recombined wavefronts are added together at the microscope analyzer. These are the colors observed for the background in the microscope viewfield either with or without a specimen positioned on the stage (Note: an optical path difference introduced by the specimen will simply add to the one created by the microscope). A path difference of 50 nanometers produces a hue between iron and lavender gray. Likewise, optical path differences of 200, 400, and 600 nanometers yield interference colors that have been named clear gray, brown-yellow, and indigo, respectively.

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