DIC Microscope Components and Imaging Mechanisms

The basic optical configuration for differential interference contrast (DIC) microscopy resembles a traditional polarized light instrument retrofitted with specialized beamsplitting (modified Wollaston or Nomarski) prisms. The relative optical orientation and sequential positioning of the DIC microscope optical components are illustrated in the tutorial, as are the wide spectrum of images obtained when the objective Nomarski prism is translated across the optical axis.

The tutorial initializes with a randomly selected specimen appearing in the DIC Specimen Image window and the Bias Retardation slider bar set to the center position. To operate the tutorial, use the mouse cursor to drag the Bias Retardation slider to the left and right. Translating the slider produces a corresponding change in the position of the objective Nomarski prism (labeled as component (6)) with respect to the optical axis of the DIC microscope. As the prism is moved to the left of the central position, the DIC specimen images goes through extinction and the amplitude profile is reversed (dark areas become light and vice versa). Translating the slider to the right of center produces a spectrum of interference colors in the specimen image to yield an effect that is commonly termed optical staining.

Strategic placement of precisely matched optical components in (or near) conjugate planes and other specific locations within the microscope is essential to the configuration scheme for differential interference contrast (see the tutorial illustration). All of the major manufacturers provide high-quality, precision DIC optical accessories, often marketed in kits, for their inverted and upright research microscopes. In general, only four basic components are required to configure a research or standard laboratory brightfield microscope for observation in differential interference contrast:

Optical path gradients in the specimen induce phase shifts in the coherent paired wavefronts sheared by the condenser prism and passing through on parallel trajectories. These phase shifts are translated into phase differences by the objective Nomarski prism, creating elliptically polarized light that is capable of passing a linear component through the analyzer and creating an image. In fact, over the entire specimen field, the presence and absence of phase gradients creates a combination of linearly and elliptically polarized wavefronts that are selectively passed by the analyzer according to the azimuths of their vibrational planes. The wavefronts that are able to pass through the analyzer are all plane-parallel and can generate an amplitude image of the specimen through interference at the image plane.

When the objective prism exactly compensates the effects of the condenser prism (as it does in Köhler illumination), the analyzer blocks wavefronts originating from all spatial locations of the field lacking phase shifts (no specimen phase gradients). The resulting background observed in the viewfield is dark (exhibiting total extinction) with the exception of regions displaying steep specimen refractive index or thickness gradients, which appear much brighter (usually in outline form). The perceived image appears very similar to images generated by the classical, and simple, darkfield illumination technique.

As the objective Nomarski prism is shifted laterally (either to the left or right of the microscope optical axis), wavefront pairs contributing to the background become increasingly retarded and out of phase with respect to one another. As a result, the degree of elliptical polarization is increased in wavefronts entering the analyzer, and the background intensity progressively transitions from black to medium and lighter shades of gray. In addition, the introduction of bias retardation shifts the position of the zeroth-order interference fringe and produces corresponding changes to the intensity levels of phase gradients in the specimen. These result in the generation of orientational-dependent bright highlights and dark shadows superimposed on the now lighter background (having a color often termed zero-order gray).

The final DIC image does not rely on the optical path difference being introduced exclusively through translation of the objective prism, and the same result can be obtained when the condenser prism is moved along the microscope optical axis. However, in most instruments it is far more convenient to produce bias retardation by shifting the position of the objective prism rather than prisms housed in a condenser turret.

When a standard objective Nomarski prism is translated along the microscope optical axis beyond path differences of one-quarter wavelength, both specimen features and the background acquire a spectrum of Newtonian interference colors similar to those observed in polarized light microscopy. The specimen and background become optically stained with a transition of color that migrates through a series of gray values through white, yellow, red-blue and higher orders. Optical staining produces dramatic and beautifully colored images, but has limited use for scientific applications. Usually, the optimum specimen contrast is limited to the range of one-twentieth to one-quarter wavelength of retardation.

Contributing Authors

Douglas B. Murphy - Department of Cell Biology and Microscope Facility, Johns Hopkins University School of Medicine, 725 N. Wolfe Street, 107 WBSB, Baltimore, Maryland 21205.

Kenneth R. Spring - Scientific Consultant, Lusby, Maryland, 20657.

Mortimer Abramowitz - Olympus America, Inc., Two Corporate Center Drive., Melville, New York, 11747.

Matthew J. Parry-Hill, Robert T. Sutter, Cynthia D. Kelly, and Michael W. Davidson - National High Magnetic Field Laboratory, 1800 East Paul Dirac Dr., The Florida State University, Tallahassee, Florida, 32310.