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.

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.

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.

Specimen Contrast in Optical Microscopy - Light can interact with a specimen through a variety of mechanisms to generate image contrast. These include reflection from the surface, absorption, refraction, polarization, fluorescence, and diffraction. Contrast can also be increased by physical modification of the microscope optical components and illumination mode, as well as manipulation of the final image through photographic or digital electronic techniques. The discussion in this section highlights various interactions between the specimen and light, and reviews some of the optical microscopy techniques that have been developed to enhance specimen contrast.

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.

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.

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 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.

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.

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.

Optical Sectioning with de Sénarmont DIC Microscopy - The ability to image a specimen in de Sénarmont DIC microscopy with large condenser and objective numerical apertures enables the creation of remarkably shallow optical sections from a focused image plane. Without the disturbance of halos and distracting intensity fluctuations from bright regions in lateral planes removed the focal point, the technique yields sharp images that are neatly sliced from a complex three-dimensional phase specimen. This property is often utilized to obtain crisp optical sections of cellular outlines in complex tissues with minimal interference from structures above and below the focal plane.

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.

Optical Sectioning in Reflected Light DIC - 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.

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.