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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.