Differential Interference Contrast
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:
- Linear Polarizer (Component 1) - Inserted into the optical pathway between the microscope light port (or anywhere after the illumination source collector lens) and the condenser lens assembly, this component is designed to produce the necessary plane-polarized light for interference imaging. The vibration plane transmission axis for the electric vector component is oriented in an East-West direction (right to left when standing in front of the microscope), typical of a standard polarized light microscope. Some differential interference contrast designs incorporate a rotating polarizer combined with a quarter-wavelength retardation plate at this position in the microscope. Together, these components are termed a de Sénarmont compensator, and are designed to provide more precise control for adjusting image contrast.
- Condenser Wollaston or Nomarski Prism (Component 2) - In order to separate the polarized light emanating from the polarizer into two components, a specialized beamsplitting prism (often referred to as the condenser prism) is placed in or near the conjugate focal plane of the condenser (Component 3) iris diaphragm aperture, as illustrated in the tutorial diagram. Incident wavefronts of plane-polarized light are split (or sheared) into mutually perpendicular (orthogonal) polarized components (termed ordinary and extraordinary wavefronts) by the Wollaston or Nomarski prism, and then pass through the specimen (Component 4).
- Objective Nomarski Prism (Component 6) - Positioned behind the objective (Component 5), either in an adjustable sliding frame or a fixed mount, a second beamsplitting prism is employed to recombine the sheared wavefronts in the conjugate plane of the objective rear aperture. This component, which is an element critical to interference and image formation, is also termed the objective prism. In most cases, the design and optical characteristics of the prisms placed in the condenser and objective focal planes differ to ensure that their interference planes coincide with optically conjugate microscope aperture planes.
- Analyzer (Component 7) - A second linear polarizer is installed behind the objective prism, usually in an intermediate tube between the microscope nosepiece and observation (eyepiece) tubes. Termed an analyzer, this polarizing element is positioned in the optical pathway before the tube lens (for infinity-corrected microscopes) and image plane. The analyzer is oriented with the transmission axis of the electric field vector perpendicular (North-South) to that of the substage polarizer. Components of circular and elliptically polarized light arriving from the objective prism pass through the analyzer and subsequently undergo interference to generate the DIC image at the microscope intermediate image plane (eyepiece fixed diaphragm or camera system projection lens aperture; Component 8).
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.
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.
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