Differential Interference Contrast
An excellent mechanism for rendering contrast in transparent specimens, differential interference contrast (DIC) microscopy is a beam-shearing interference system in which the reference beam is sheared by a minuscule amount, generally somewhat less than the diameter of an Airy disk. The technique produces a monochromatic shadow-cast image that effectively displays the gradient of optical paths for both high and low spatial frequencies present in the specimen. Those regions of the specimen where the optical paths increase along a reference direction appear brighter (or darker), while regions where the path differences decrease appear in reverse contrast. As the gradient of optical path difference grows steeper, image contrast is dramatically increased.
Brief Overview of DIC Microscopy - In the mid-1950s, a French optics theoretician named Georges Nomarski modified the Wollaston prism used for detecting optical gradients in specimens and converting them into intensity differences. Today there are several implementations of this design, which are collectively called differential interference contrast (DIC). Living or stained specimens, which often yield poor images when viewed in brightfield illumination, are made clearly visible by optical rather than chemical means.
Fundamental Concepts in DIC Microscopy - Through a mechanism quite different from phase contrast, differential interference contrast converts specimen optical path gradients into amplitude differences that can be visualized as improved contrast in the resulting image. The optical components required for differential interference contrast microscopy do not mask or otherwise obstruct the objective and condenser diaphragms (as in phase or Hoffman modulation contrast), thus enabling the instrument to be employed at full numerical aperture. The result is a dramatic improvement in resolution (particularly in the direction of the optical axis), elimination of halo artifacts, and the ability to produce excellent images with relatively thick specimens. In addition, differential interference contrast produces an image that can be easily manipulated using digital and video imaging techniques to further enhance contrast.
de Sénarmont Bias Retardation in DIC Microscopy - In traditional differential interference contrast (DIC) microscope system designs, bias retardation is introduced into the optical train by translating one of the matched (condenser and objective) Nomarski or modified Wollaston prisms across the optical axis of the microscope to produce a constant optical path difference. The same effect can also be achieved through the application of a fixed Nomarski prism system and a simple de Sénarmont compensator consisting of a quarter-wavelength retardation plate in conjunction with either the polarizer or analyzer.
DIC Microscope Configuration and Alignment - Differential interference contrast (DIC) optical components can be installed on virtually any brightfield transmitted, reflected, or inverted microscope, provided the instrument is able to accept polarizing filters and the specially designed condenser and objective prisms (together with the housings) necessary to perform the technique. All of the major microscope manufacturers produce DIC accessories for their research-level microscopes, and these are often bundled together as matched kits containing all of the required hardware and optical components. In the standard configuration, a differential interference contrast microscope contains the polarizing elements typically encountered on a polarized light microscope and, in addition, two specially constructed birefringent compound prisms. Termed Wollaston or Nomarski prisms, these optical beamsplitters (and beam combiners) are positioned to project interference patterns of sheared wavefronts into the condenser front focal plane and the objective rear focal plane.
de Sénarmont DIC Microscope Configuration - Configuration of either a transmitted or reflected optical microscope for operation in differential interference contrast (DIC) using a de Sénarmont compensator offers far more latitude and accuracy for the introduction of bias retardation than is possible with systems that rely on translation of the objective Nomarski (or Wollaston) prism across the optical pathway. Virtually any microscope that contains polarizing elements and the necessary condenser and objective beamsplitting compound prisms can be easily converted for operation in de Sénarmont mode, regardless of whether the microscope was originally designed for this purpose.
Comparison of Phase Contrast and DIC Microscopy - Phase contrast and differential interference contrast (DIC) microscopy are complementary techniques capable of producing high contrast images of transparent biological phases that do not ordinarily affect the amplitude of visible light waves passing though the specimen. The most fundamental distinction between differential interference contrast and phase contrast microscopy is the optical basis upon which images are formed. Phase contrast yields image intensity values as a function of specimen optical path length magnitude, with very dense regions (those having large path lengths) appearing darker than the background. The situation is quite distinct for differential interference contrast, where optical path length gradients (in effect, the rate of change in the direction of wavefront shear) are primarily responsible for introducing contrast into specimen images.
Reflected Light DIC Microscopy - Reflected light microscopy is one of the most common techniques applied in the examination of opaque specimens that are usually highly reflective and, therefore, do not absorb or transmit a significant amount of the incident light. Slopes, valleys, and other discontinuities on the surface of the specimen create optical path differences, which are transformed by reflected light DIC microscopy into amplitude or intensity variations that reveal a topographical profile. Unlike the situation with transmitted light and semi-transparent phase specimens, the image created in reflected light DIC can often be interpreted as a true three-dimensional representation of the surface geometry, provided a clear distinction can be realized between raised and lowered regions in the specimen.
Fluorescence and DIC Combination Microscopy - Fluorescence microscopy can be combined with contrast enhancing techniques, such as differential interference contrast (DIC) and phase contrast illumination, to minimize the effects of photobleaching. A specific area of interest can be located by examining the specimen in either DIC or phase contrast, and then switching the microscope to fluorescence mode without relocating the specimen. In addition, utilizing DIC to image specimens in combination with fluorescence excitation can be of significant value in determining the precise location of fluorescent species.
Georges Nomarski (1919-1997) - A Polish born physicist and optics theoretician, Georges Nomarski adopted France as his home after World War II. Nomarski is credited with numerous inventions and patents, including a major contribution to the well-known differential interference contrast (DIC) microscopy technique. Also referred to as Nomarski interference contrast (NIC), the method is widely used to study live biological specimens and unstained tissues.
Henri Hureau de Sénarmont (1808-1862) - Sénarmont was a professor of mineralogy and director of studies at the École des Mines in Paris, especially distinguished for his research on polarization and his studies on the artificial formation of minerals. He also contributed to the Geological Survey of France by preparing geological maps and essays. Perhaps the most significant contribution made by de Sénarmont to optics was the polarized light retardation compensator bearing his name, which is still widely utilized today.
William Hyde Wollaston (1766-1828) - Although formally trained as a physician, Wollaston studied and made advances in many scientific fields, including chemistry, physics, botany, crystallography, optics, astronomy and mineralogy. He is particularly noted for originating several inventions in optics, including the Wollaston prism that is fundamentally important to interferometry and differential interference (DIC) contrast microscopy.
Robert Day Allen (1927-1986) - Robert Day Allen was a renowned microscopist, a prominent researcher of cell motility processes, and a co-developer of video-enhanced contrast microscopy ((VEC)), which is a modification of the traditional form of differential interference contrast (DIC) microscopy. Along with Georges Nomarski and G. B. David, Allen assisted the Carl Zeiss Optical Company in developing a Nomarski differential interference microscope for transmitted light applications. In a hallmark paper published in Zeitschrift für wissenschaftliche Mikroskopie und mikroskopische Technik, Allen and his colleagues defined the basic principles of the DIC technique and the interpretation of images.
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.
Optical Path Gradients and Amplitude Profiles - The difference in optical path experienced by orthogonal wavefronts passing through a specimen in differential interference contrast (DIC) microscopy is converted by the optical system to a change in amplitude in the final image observed in the eyepieces. This interactive tutorial explores the relationship between optical path gradients and amplitude (intensity) profiles for a variety of semi-transparent specimens.
Origin and Variation of Image Contrast - Image intensity in differential interference contrast (DIC) microscopy is a function of the difference in optical path experienced by the extraordinary and ordinary wavefronts as they travel through phase gradients in the specimen. This interactive tutorial explores how variations in the level of bias retardation introduced by a Nomarski or Wollaston prism affect the optical path difference and resulting specimen intensity in a DIC microscope.
Bias Retardation Effects on Specimen Contrast - The introduction of bias retardation in differential interference contrast (DIC) microscopy renders the specimen image in pseudo three-dimensional relief where regions of increasing optical path length (sloping phase gradients) appear much brighter (or darker), and those exhibiting decreasing path length appear in reverse. This interactive tutorial explores the effects of varying bias retardation on contrast as a function of thickness for a wide spectrum of semi-transparent specimens.
The Interference Background Image - 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.
Specimen Orientation Effects on DIC Images - In differential interference contrast images, shadow and highlight intensity (amplitude) is greatest along the shear axis of the microscope, and regions of constant refractive index display intensity values that are identical to that of the background. This interactive tutorial explores how image amplitude fluctuates as the specimen orientation is varied with respect to the microscope shear axis.
Wavefront Shear in Wollaston and Nomarski Prisms - A Wollaston prism is composed of two geometrically identical wedges of quartz or calcite (which are birefringent, or doubly-refracting materials) cut in a way that their optical axes are oriented perpendicular when they are cemented together to form the prism. If a linear polarizer is oriented so that plane-polarized light enters the prism at a 45-degree angle with respect to the optical axes of the two birefringent prism halves, the light is sheared into two plane-polarized components that are oriented mutually perpendicular to each other. This interactive tutorial examines differences between the location of the interference plane in Wollaston and Nomarski prisms, and how the position of the plane can be varied with changes to the optical axis orientation in a single prism wedge.
Nomarski Prism Action in Polarized Light - When a Nomarski or modified Wollaston compound differential interference contrast (DIC) prism is sandwiched between two crossed polarizers and examined with light transmitted through both polarizers and the prism, a pattern of parallel interference fringes with a predominant central black band (fringe) can be observed. This interactive tutorial explores how varying prism wedge geometry, utilized for different objective numerical apertures, affects the interference pattern observed between crossed polarizers.
DIC Wavefront Relationships and Image Formation - The spatial relationship and phase difference between ordinary and extraordinary wavefronts in differential interference contrast (DIC) microscopy is a primary factor in determining how image formation occurs. This interactive tutorial explores wavefront relationships in the DIC microscope optical train, and how these relationships affect image formation.
Optical Staining with DIC Microscopy - By introducing birefringent compensator plates into the optical pathway of a differential interference contrast (DIC) microscope, transparent specimens that are otherwise rendered over a limited range of grayscale values can be transformed to display a wide array of colors through the technique known as optical staining. This interactive tutorial explores how varying the amount of bias retardation can affect the appearance and level of staining achieved in the specimen image.
Optical Sectioning in DIC Microscopy - The ability to image a specimen in differential interference contrast (DIC) microscopy with large condenser and objective numerical apertures enables the creation of optical sections from a focused image that are remarkably shallow. Without the disturbance of halos and distracting intensity fluctuations from bright regions in axial planes removed from the focal point, the technique yields sharp images that are neatly sliced from a complex three-dimensional phase specimen. This interactive tutorial explores optical sectioning in DIC microscopy utilizing a wide spectrum of specimens having varied thickness. A similar tutorial on MicroscopyU reviews the technique of optical sectioning with a de Sénarmont DIC microscope.
DIC Microscope Component Alignment - The proper adjustment and alignment of differential interference contrast (DIC) optical components is critical to imaging performance, so it is imperative that the microscopist recognize misalignments and component mismatches, and take the necessary steps to correct these errors. This interactive tutorial, hosted on the Nikon MicroscopyU website, examines conoscopic and orthoscopic viewfields in a DIC microscope under a variety of configurational motifs, and discusses many of the important aspects recommended for satisfactory microscope alignment.
Comparison of Phase Contrast and DIC Microscopy - The most fundamental distinction between differential interference contrast (DIC) and phase contrast microscopy is the optical basis upon which images are formed by the complementary techniques. Specimens examined by these contrast-enhancing methods produce images that are often quite different in appearance and character when objectively compared. This MicroscopyU interactive tutorial explores many of the similarities and differences exhibited between images captured with phase contrast and DIC microscopy.
Optical Sectioning with Phase Contrast and DIC - One of the primary advantages of differential interference contrast (DIC) microscopy over phase contrast is the ability to utilize the instrument at full numerical aperture without suffering the masking effects of phase plates or condenser annuli, which severely restrict the size of the condenser and objective apertures. The major benefit is improved axial resolution, particular with respect to the ability of the DIC microscope to produce excellent high-resolution images at large aperture sizes. This interactive tutorial explores and compares optical sectioning of thick specimens with DIC and phase contrast, and reveals the benefits of unrestricted aperture effects on obtaining well-defined sections.
de Sénarmont Compensators - A de Sénarmont compensator is composed of a linear polarizer combined with a quarter-wavelength retardation plate, and is capable of producing either linear, elliptical, or circularly polarized light, depending upon the orientation of the polarizer vibration axis with respect to the fast and slow axes of the retardation plate. This interactive tutorial explores the relationship between wavefronts emanating from the compensator as the polarizer is rotated through its useful range.
DIC Microscopy with de Sénarmont Compensators - Although in traditional designs, differential interference contrast (DIC) microscopes introduce bias retardation into the matched condenser and objective Nomarski (or Wollaston) prisms by translating one of the prisms across the optical axis, the same effect can also be achieved through the use of a simple de Sénarmont compensator with fixed Nomarski prisms. This MicroscopyU interactive tutorial examines the relationship between wavefronts emerging from a de Sénarmont compensator and how they can be controlled to produce positive and negative bias retardation (contrast) effects in a DIC microscope.
Wavefront Relationships in de Sénarmont and Nomarski DIC - In differential interference contrast (DIC) microscopy, the spatial relationship and phase difference between ordinary and extraordinary wavefronts is governed either by the position of the objective prism (Nomarski DIC) or the relationship between the polarizer and a thin quartz retardation plate in a de Sénarmont design. This interactive tutorial explores the similarities and differences between the wavefront relationship in the two microscope configurations.
Wavefront Fields in DIC Microscopy - Wavefront fields traversing the optical train of a differential interference contrast (DIC) microscope undergo several reorientations as they encounter various polarizing, phase retarding, and beamsplitting elements present in the system. Linearly polarized light emerging from the polarizer is separated into orthogonal components upon entering the birefringent condenser Wollaston (or Nomarski) prism and is then sheared at the boundary between the prism wedges. This interactive tutorial explores the wavefront relationships involving polarized and orthogonal wavefront components in both de Sénarmont and traditional Nomarski optical configurations.
The de Sénarmont DIC Microscope Optical Train - Although traditional differential interference contrast (DIC) optical systems introduce bias retardation into the wavefront field by translation of the objective Nomarski prism, the same effect can be achieved through the application of a fixed Nomarski (or Wollaston) prism system and a simple de Sénarmont compensator consisting of a quarter-wavelength retardation plate in conjunction with either the polarizer or analyzer. This interactive tutorial explores the wavefront relationship in a de Sénarmont DIC microscope optical train as the polarizer is rotated with respect to the fast axis of the retardation plate.
Wavefront Relationships in Reflected Light DIC Microscopy - In reflected light differential interference contrast (DIC) microscopy, the spatial relationship and phase difference between ordinary and extraordinary wavefronts passing through the optical system is governed either by the position of the objective prism (Nomarski DIC) or the orientational relationship between the polarizer and a thin quartz retardation plate in a de Sénarmont design. This interactive tutorial explores the similarities and differences between the wavefront relationships in the two microscope configurations.
Reflected DIC Optical Sectioning - The ability to capitalize on large objective numerical aperture values in reflected light DIC microscopy enables the creation of optical sections from a focused image that are remarkably shallow. Without the confusing and distracting intensity fluctuations from bright regions occurring in optical planes removed from the focal point, the technique yields sharp images that are neatly sliced from a complex three-dimensional opaque specimen having significant surface relief. This property is often employed to obtain crisp optical sections of individual features on the surface of integrated circuits, as explored in the interactive tutorial, with minimal interference from obscuring structures above and below the focal plane. A similar tutorial on MicroscopyU reviews the technique of optical sectioning with a different set of integrated circuits.
Digital Image Galleries
Differential Interference Contrast Digital Image Gallery - Thin unstained, transparent specimens are excellent candidates for imaging with classical differential interference (DIC) microscopy techniques over a relatively narrow range (plus or minus one-quarter wavelength) of bias retardation. The digital images presented in this gallery represent a wide spectrum of specimens, which vary from unstained cells, tissues, and whole organisms to both lightly and heavily stained thin and thick sections. In addition, several specimens exhibiting birefringent character are included to demonstrate the kaleidoscopic display of color that arises when anisotropic substances are imaged with this technique.
Phase Contrast and DIC Comparison Image Gallery - Phase contrast and differential interference contrast (DIC) should be considered as complementary (rather than competing) techniques, and employed together to fully investigate specimen optical properties, dynamics, and morphology. In many cases, each technique will reveal specific details about a particular specimen that is not apparent from observing images captured by other methods. The wide variety of images presented in this MicroscopyU gallery are derived from both thick and thin transparent specimens, as well as specimens that have inherent contrast originating from synthetic dyes (stains) or natural pigments.
Selected Literature References and Glossary of Terms
Glossary of Common Terms in DIC Microscopy - The complex nomenclature of differential interference contrast 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.
Selected DIC Microscopy Literature References - A number of review articles on differential interference contrast (DIC) microscopy have been published by leading researchers in the field, and were utilized as references to prepare discussions included in the Molecular Expressions Microscopy Primer. This section contains periodical location information about these articles, as well as providing a listing of selected original research reports and books describing specimen contrast and the classical techniques of differential interference contrast light microscopy.
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.
Edward D. Salmon - Department of Cell Biology, The University of North Carolina, Chapel Hill, North Carolina 27599.
Kenneth R. Spring - Scientific Consultant, Lusby, Maryland, 20657.
Chris Brandmaier, Mel Brenner, and Stanley Schwartz - Industrial and Bioscience Departments, Nikon Instruments, Inc., 1300 Walt Whitman Road, Melville, New York 11747.
H. Ernst Keller - Carl Zeiss Inc., One Zeiss Dr., Thornwood, NY, 10594.
Mortimer Abramowitz - Olympus America, Inc., Two Corporate Center Drive., Melville, New York, 11747.
Maksymilian Pluta - Physical Optics Department, Institute of Applied Optics, 18 Kamionkowska Street, Warsaw, Poland, 03-805.
Matthew Parry-Hill, Robert T. Sutter, Thomas J. Fellers, 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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