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Differential Interference Contrast

Glossary of Common Terms in DIC Microscopy

The complex nomenclature of differential interference contrast (DIC) microscopy is often confusing to both beginning students and seasoned research microscopists alike. Because this contrast enhancing technique relies so heavily on polarized light, and the separation, retardation, recombination, and interaction of mutually perpendicular wavefronts, many of the rather numerous common terms in the field have multifaceted, underlying, or implied definitions. This resource is provided as a guide and reference tool for visitors who are exploring the large spectrum of specialized topics in DIC microscopy.

Analyzer - A linear polarizing element employed to analyze or determine the electric vector vibration plane of an incident wavefront of polarized light. In DIC microscopy, the analyzer is positioned behind the objective and Nomarski prism in the imaging pathway, and passes only the linear components in a single orientation emerging from the optical system.

Anisotropic - Materials that are anisotropic have non-uniform spatial distribution of optical properties (for example, refraction, transmission, reflection). In polarized light and DIC microscopy, anisotropy refers to the preferential orientation of optical properties with respect to the vibration plane of linearly polarized light.

Azimuth Angle - Azimuth is a term used to describe the orientation of a specimen in the imaging plane (stage) of a DIC microscope. On a graph having polar coordinates graduated in 360 degrees, the azimuth angle is subtended between a fixed reference (designated as zero degrees) and a vector rotated about the origin. The term is also frequently employed to describe the direction(s) of illumination of wavefronts emanating from a restricted aperture.

Bertrand Lens - A telescopic lens system assembled into the observation tubes of an optical microscope that is utilized to observe the objective rear focal plane (aperture). In DIC microscopy, the Bertrand lens is often employed to align the microscope, set the condenser aperture diaphragm size, and to ensure that the interference fringes of Wollaston and Nomarski prisms are oriented correctly for maximum overlap.

Bias Retardation - The alteration of optical path differences between orthogonal wavefronts produced by adjusting the position of a Wollaston or Nomarski prism, or by rotating the polarizer in a de SÚnarmont compensator. In DIC microscopy, bias retardation is introduced by the operator to increase or decrease the contrast of a specimen.

Birefringence - The phenomenon of double refraction of light wavefronts in a transparent, molecularly ordered material produced by the existence or orientation-dependent differences in refractive index. The term birefringence also commonly refers to the refractive index difference experienced by a transmitted wave through such a material. Wavefronts of light incident on a birefringent specimen are split into ordinary and extraordinary components that can recombine after emergence from the specimen to produce linearly, elliptically, or circularly polarized light.

Circularly Polarized Light - A common form of polarized light in which the electric vector of the wave rotates around the axis of propagation of the wavefront, thus sweeping out a circular spiral. Two coherent and coaxial orthogonal wavefronts that have a phase difference of 90 degrees produce circularly polarized light when the individual electric vector components of each wavefront are summed.

Coherent Light - A beam of light composed of wavefronts that are vibrating in the same phase, although not necessarily lying in the same plane of vibration. To maintain a common phase over long distances, coherent wavefronts must be monochromatic (have the same wavelength). As an example, most laser sources produce monochromatic, linearly polarized, highly coherent wavefronts.

Compensator - A birefringent slab of optical quartz, mica, or similar material that is positioned almost anywhere between the polarizer and analyzer in a polarized microscope, but limited to the exterior of the prisms and other imaging components of a DIC microscope. The slab can be tilted and/or rotated to achieve varying optical effects. A compensator changes the optical path difference between orthogonal wavefronts, and can be utilized to perform quantitative measurements of the relative ordinary and extraordinary wavefront retardations, or for qualitative purposes in adjusting image contrast and brightness. Compensator plates are often used in DIC microscopy for optical staining of transparent specimens.

Constructive Interference - In the field of wave optics and image formation, constructive interference occurs when the summation of electric vectors from constituent waves results in an amplitude greater than that of the individual components. For any form of interference to take place, the electric vector component of each wave involved must be vibrating in a common plane.

Contrast - Qualitatively defined as the perceived difference in brightness (intensity or irradiance) between a specimen and its surrounding medium, optical contrast is also more quantitatively described as the ratio of the light intensity of a specimen to the light intensity of the background.

de SÚnarmont Compensation - In polarized light and DIC microscopy, de SÚnarmont compensation utilizes a fixed quarter-wavelength retardation plate together with a rotating polarizer (or analyzer) as a method for measuring or creating optical path differences between orthogonal components. In addition, de SÚnarmont compensation is used to introduce contrast into a DIC specimen image by varying the amount of bias retardation applied to the optical system. Retardation values ranging between one-twentieth and a full wavelength can be measured with an accuracy of 0.15 nanometers.

Destructive Interference - In the field of wave optics and image formation, destructive interference occurs when the summation of electric vectors from constituent waves results in an amplitude less than that of the individual components. For any form of interference to take place, the electric vector component of each wave involved must be vibrating in a common plane.

Differential Interference Contrast (DIC) Microscopy - A popular mode of optical microscopy that utilizes dual-beam interference optics to transform localized gradients in optical path length into regions of contrast in the image of a specimen. The technique is also commonly referred to as Nomarski DIC to honor the name of an inventor, Georges Nomarski. In DIC microscopy, the specimen is illuminated by a beam of closely spaced coherent orthogonal wavefront pairs that are generated by a beamsplitter termed a Wollaston or Nomarski prism. Members of the wavefront pairs experience different optical path lengths if they traverse a gradient in refractive index or geometrical thickness in a phase specimen. Optical path differences become translated into amplitude differences (contrast) having a characteristic relief-like shadow-cast appearance when the wavefronts recombine to form an image.

Double Refraction - In polarized and DIC optical microscopy, double refraction (or birefringence) is defined as the splitting of light into distinct orthogonal ordinary and extraordinary wavefronts in a birefringent material (such as an anisotropic specimen or a Wollaston prism). When a birefringent calcite crystal is placed onto a page of printed words, the effects of double refraction are clearly observed as an overlapping, double image of the text.

Elliptically Polarized Light - A common form of polarized light in which the electric vector of the wave rotates around the axis of propagation of the wavefront, thus sweeping out an elliptical spiral. Two coherent and coaxial orthogonal wavefronts that have a phase difference greater than zero but less than 90 degrees produce elliptically polarized light when the individual electric vector components of each wavefront are summed.

Extinction - The term refers to blockage of light transmission through an analyzer (polarizer) in polarized and DIC optical microscopy. This condition occurs when the vibrational plane of the electric vector in a linearly polarized wavefront is oriented perpendicularly with respect to the transmission axis of the analyzer. If two Polaroid filters are held together with their transmission axes crossed, extinction is said to occur when the magnitude of light transmission drops to a minimum. The extinction factor is defined as the ratio of light amplitudes when the electric vector of a linearly polarized wavefront and the transmission axis of the analyzer are parallel (maximum transmission) and crossed (extinction or minimum transmission).

Extraordinary Wave - Also termed a wavefront or ray, the extraordinary component of linearly polarized light that is transmitted through a birefringent medium traveling with a velocity that varies with the direction of transmission. The extraordinary wave is oriented parallel to the optical axis of a quartz wedge as it travels through a Wollaston or Nomarski prism. An extraordinary wavefront emanating from an imaginary point source in a birefringent medium can be described as the surface of a three-dimensional ellipsoid.

Fast Axis - In polarized light and DIC microscopy, the fast axis is defined as the long axis of the wavefront ellipsoid, a construction utilized to describe the surface of an emergent wavefront from a point source of light in a birefringent material. The fast axis indicates the direction of low refractive index in a birefringent specimen.

Interference - The summation of two or more interacting electromagnetic waves is commonly known as interference between the waves. Two light waves can only interfere if a component of the electric vector from each wave vibrates in a common plane. Resultant waves with amplitudes greater or less than the constituent waves are said to represent constructive and destructive interference, respectively.

Isotropic - A general term used to describe the uniform spatial distribution of optical characteristics displayed by a specimen or propagation medium. Isotropic specimens have identical properties when probed from any direction. In polarized light and DIC microscopy, isotropic specimens possess a uniformity of optical properties with respect to the vibration plane of polarized light.

Linearly Polarized Light - A wavefront in which the electric vectors of the constituent components vibrate in planes that are mutually parallel. Linearly polarized light does not necessarily have coherent or monochromatic (single wavelength) characteristics.

Nomarski Prism - Similar in construction to a Wollaston prism, the Nomarski beamsplitter prism design incorporates a specially cut quartz wedge having the optical axis oriented obliquely to the flat face of the lower prism wedge element. The angle of the optical axis dictates where the interference plane of the prism is located (usually at some point on the exterior of the prism).

Objective Rear Focal Plane - An illumination aperture conjugate plane in the optical microscope that is located within or near the rear lens element of the objective, and is the site of diffraction image formation. In a DIC microscope, a Nomarski or Wollaston prism is located at or near this plane.

Optical Axis - As applied to birefringent crystals in polarized light and DIC microscopy, the path followed by the ordinary wavefront in a birefringent material. Aligned optical systems possess an optical axis consisting of a straight line joining the centers of curvature of lens surfaces contained within the system.

Optical Path Difference - The difference in optical path length, abbreviated as OPD, between two coherent (often orthogonal) wavefronts due to variations in geometrical length and/or refractive index. In interference optics, optical path length differences are utilized to determine the relative phase shift and the degree of interference between zeroth-order and higher-order diffracted waves that have common origins. The measurement of an optical path difference is often expressed in units of wavelength or fractions of a wavelength.

Optical Path Length - Defined as the product of the optical path geometrical length and the refractive index in a homogeneous medium through which a wavefront traverses. Optical path length is usually expressed in units of time or as a fraction or multiple of a given wavelength. In media that are inhomogeneous (multiple domains), the optical path length is the sum or integral of the product resulting from the geometrical lengths and refractive indices of all parts.

Orthogonal Wavefronts - Two closely spaced coherent wavefronts of light that are oriented with the electric vector vibrations perpendicular to one another. The wavefronts may be coaxial or separated by a shear distance.

Ordinary Wave - Also termed a wavefront or ray, the ordinary component of linearly polarized light that is transmitted through a birefringent medium traveling with a velocity that does not vary with direction. An ordinary wave obeys normal laws of refraction. The ordinary wave is oriented perpendicular to the optical axis of a quartz wedge as it travels through a Wollaston or Nomarski prism.

Phase - A description for the relative position in a cyclical or wave motion. Because one complete wavelength is described as 2p radians or 360 degrees, the phase of a wave is given in radians, degrees, or fractions of a wavelength. The term in phase refers to phase angles between two wavefront occurrences of zero and 360 degrees or a whole number multiple of these.

Phase Difference - The phase angle by which one periodic disturbance or wavefront lags behind or precedes another in time or space. Phase differences are usually described in terms of fractions or multiples of a wavelength.

Phase Gradient - In DIC microscopy, the gradient of phase shifts in an image corresponding to actual optical path differences in the specimen. Phase gradients are primarily responsible for generation of contrast when bias retardation is applied to a DIC optical system.

Phase Specimen - Specimens that shift the phase of light waves traveling through without seriously affecting the amplitude. Many phase specimens are completely or partially transparent, and are ideal candidates for imaging with DIC microscopy techniques.

Plane Parallel - A common term that is widely utilized in wave optics to describe waves vibrating in a plane that is parallel to some reference plane, but not necessarily in the reference plane itself. Linearly polarized light is referred to as being plane parallel.

Polar - Linear polarizing film (such as a dichroic or Polaroid filter) is often referred to as a polar, particular when utilized as a polarizer or analyzer in producing and analyzing polarized light in DIC microscopy.

Polarization Cross - In polarized light and DIC microscopy, the polarization cross appears as a dark (almost black) upright Maltese cross in the rear aperture of the objective under conditions of extinction when the polarizer and analyzer have crossed transmission axes. Ideally, the rear aperture is uniformly dark under these conditions, but the depolarization of light by curved lens surfaces of the condenser and objective produces brightness in four quadrants, hence the appearance of a cross.

Polarized Light - This commonly used term refers to light waves whose electric vectors vibrate in a plane-parallel orientation at any point along the axis of propagation. Polarized light can be linearly polarized where vibrations at all locations are plane parallel, or it can be elliptically or circularly polarized with vibration axes that vary depending on location along the axis of propagation. Polarized light is not necessarily monochromatic (single wavelength) or coherent.

Polarizer - An anisotropic optical device that receives random light and transmits linearly polarized light. In DIC microscopy, polarizers are often fabricated from sheets of oriented dichroic molecules (Polaroid filters) or from slabs of birefringent crystalline materials.

Polaroid Filter - A sheet of aligned long-chain polyvinyl alcohol molecules impregnated with ordered microcrystals of polyiodide. The electric vectors of incident wavefronts vibrating along the axis parallel to the crystalline axes are absorbed and removed, resulting in the transmission of waves that are linearly polarized.

Refractive Index (Wavefront) Ellipsoid - Being the figure of revolution of an ellipse, an ellipsoid, when rotated around the major axis, is utilized to describe the surface wavefront locations of extraordinary waves propagating outward from a central point through a birefringent material. The same type of figure is employed to describe the orientation and magnitude of the two extreme refractive index values that exist in birefringent uniaxial crystals and ordered biological materials.

Relative Retardation - The relative phase shift between two wavefronts, usually expressed in fractions of a wavelength. Retardation between wavefronts is critical to the introduction of specimen contrast in DIC microscopy.

Retardation Plate - A birefringent plate, positioned between the polarizer and analyzer in a polarized light or DIC microscope, which introduces a relative retardation between orthogonal wavefronts to yield a change in image contrast or to render optical path differences in Newtonian interference colors. In polarized light microscopy, the retardation plate produces a shift that is added to that induced by a birefringent specimen. When employed in a microscope to measure the amount of relative retardation by the specimen, the plate is termed a compensator.

Shear - Angular splitting of coaxial orthogonal wavefronts resulting in a spatial separation defined by a specific angle (the shear angle). Shear occurs at the refractive index junction between cemented quartz wedges in a Wollaston prism or at the glass-air interface in the lower quartz wedge in a Nomarski prism.

Shear Angle - The specific angle defined by angular separation of paraxial orthogonal wavefronts as they become displaced and spatially separated in a Wollaston or Nomarski prism.

Shear Axis - The principal axis of a Wollaston or Nomarski prism lying parallel to the optical axis of the lower quartz wedge in each of these compound assemblies. The shear axis defines the direction of wavefront displacement of polarized light passing through the prism. In a DIC image, the shear axis is identified by an imaginary line connecting the brightest and darkest shaded edges of refractile specimen features.

Shear Distance - Displaced orthogonal ordinary and extraordinary wavefronts are separated by a spatial separation length, the shear distance, derived from the geometrical constraints of the Wollaston or Nomarski prism housed in the microscope condenser. The shear distance ranges between 0.1 and 1.5 micrometers, a linear range that is designed to be slightly smaller than (or in some cases equal to) the lateral resolution of the objective.

Slow Axis - Defined as the short axis of the wavefront ellipsoid, which is the construction employed to describe the surface of an emergent wavefront from a point source of light in a birefringent material. The slow axis indicates the direction of high refractive index in a specimen.

Video-Enhanced Contrast - Often referred to with the acronym VEC-DIC (video-enhanced contrast DIC), the term describes a method of image (contrast) enhancement in which the offset and gain positions of the camera and/or image processor are adjusted close together to include the gray-level values in a feature of interest. As a result, the specimen image is displayed at very high contrast, rendering visible features that can be difficult (or impossible) to distinguish when the image is displayed with a larger dynamic range.

Wollaston Prism - A specialized compound prism beamsplitter fabricated from two wedge-shaped slabs of high-grade optical quartz (a uniaxial birefringent crystal), which are cut with the optical axes oriented at right angles (perpendicular) to each other. The wedges are cemented together at the hypotenuse to generate an optically anisotropic compound plate where the axis of the first wedge is perpendicular to the optical axis of the second wedge. In DIC microscopy, specimens are probed by pairs of closely spaced orthogonal waves of linearly polarized light that are generated by a Wollaston prism. An important feature of the prism is the interference plane, which lies inside the prism.

Contributing Authors

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

Jan Hinsch - Leica Microsystems, Inc., 90 Boroline Road, Allendale, New Jersey, 07401.

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

Michael W. Davidson - National High Magnetic Field Laboratory, 1800 East Paul Dirac Dr., The Florida State University, Tallahassee, Florida, 32310.


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