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.

Interactive Tutorials

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.

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.

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.

Differential Interference Contrast

Introduction

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.

In transmitted light DIC, light from the lamp is passed through a polarizer located beneath the substage condenser, in a manner similar to polarized light microscopy. Next in the light path (but still beneath the condenser) is a modified Wollaston prism that splits the entering beam of polarized light into two beams traveling in slightly different directions. The Wollaston prism is composed of two quartz wedges cemented together, from which emerging light rays vibrate at 90 degrees relative to each other with a slight path difference. A different Wollaston prism is needed for each objective of different magnification. Wollaston prisms are usually loaded into a revolving turret on the condenser, which allows the microscopist to rotate the appropriate prism into the light path when changing magnifications.

Interactive Tutorial

Wollaston Prism Wavefronts

Examine how a Wollaston prism acts to split or shear a polarzied beam of light into an ordinary and an extraordinary ray oriented in mutually perpendicular directions.

The plane-polarized light, vibrating only in one direction (East-West) perpendicular to the propagation direction of the light beam, enters the beam-splitting modified Wollaston prism and is split into two rays, vibrating perpendicular to each other. These two rays travel close together but in slightly different directions (see Figure 2). The rays intersect at the front focal plane of the condenser, where they pass traveling parallel and extremely close together with a slight path difference, but they are vibrating perpendicular to each other and are therefore unable to cause interference. The distance between the rays, called the shear, is so minute that it is less than the resolving ability of the objective.

The split beams enter and pass through the specimen where their wave paths are altered in accordance with the specimen's varying thickness, slopes, and refractive indices. These variations cause alterations in the wave path of both beams passing through areas of any specimen details lying close together. When the parallel beams enter the objective, they are focused above the rear focal plane where they enter a second modified Wollaston prism that combines the two beams at a defined distance outside of the prism itself. This removes the shear and the original path difference between the beam pairs. As a result of having traversed the specimen, the paths of the parallel beams are not of the same length (optical path difference) for differing areas of the specimen.

In order for the beams to interfere, the vibrations of the beams of different path length must be brought into the same plane and axis. This is accomplished by placing a second polarizer (analyzer) above the upper Wollaston beam-combining prism. The light then proceeds toward the eyepiece where it can be observed as differences in intensity and color. The design results in one side of a detail appearing bright (or possibly in color) while the other side appears darker (or another color). This shadow effect bestows a pseudo three-dimensional appearance to the specimen.

In some microscopes, the upper modified Wollaston prism is combined in a single fitting with the analyzer incorporated above it. The upper prism may also be arranged so it can be moved horizontally. This allows for varying optical path differences by moving the prism, providing the user a mechanism to alter the brightness and color of the background and specimen. Because of the prism design and placements, the background will be homogeneous for whatever color has been selected.

Interactive Tutorial

DIC Microscope Components and Imaging Mechanisms

Explore how changing the bias in the upper Wollaston prism affects image contrast in the DIC microscope.

The color and/or light intensity effects shown in the image are related especially to the rate of change in refractive index, specimen thickness, or both. Orientation of the specimen can have pronounced effect on the relief-like appearance and often, rotation of the specimen by 180 degrees changes a hill into a valley or visa versa. The three-dimensional appearance is not a representation of the true geometric nature of the specimen, but is an exaggeration based on optical thickness. It is not suitable for accurate measurement of actual heights and depths.

There are numerous advantages in DIC microscopy as compared to phase contrast microscopy. With DIC, it is possible to make fuller use of the numerical aperture of the system and to provide optical staining (color). DIC also allows the microscope to achieve excellent resolution. Use of full objective aperture enables the microscopist to focus on a thin plane section of a thick specimen without confusing images from above or below the plane. Annoying halos, often encountered in phase contrast, are absent in DIC images, and suitable achromat and fluorite objectives can be used for this work.

A downside is that plastic tissue culture vessels and other birefringent specimens yield confusing images in DIC, hence Hoffman modulation contrast is recommended for tissue culture work. Also, high-quality apochromatic objectives are now designed to be suitable for DIC. Figure 3 presents transmitted and reflected light DIC photomicrographs of the mouthparts of a blowfly (transmitted DIC; Figure 3(a)) and surface defects in a ferro-silicate alloy (reflected DIC; Figure 3(b)). Both photomicrographs were made using a retardation plate with a 10x objective.

The equipment needed for DIC microscopy includes a polarizer, a beam-splitting modified Wollaston prism below the condenser, a beam-recombining modified Wollaston prism above the objective, and an analyzer above this upper prism. Individual prisms are required (for each objective) below the condenser (Figure 4). For the upper prism, a single prism may serve for all objectives. The upper prism can be moved laterally.

The distance of the placement of the lower prism in relation to the front focal plane of the condenser and the distance of the upper prism from the back focal plane of the objective are quite critical. Manufacturers therefore designate which of their objectives are suitable for their particular DIC apparatus.

The DIC condenser contains four or more prisms, a brightfield opening with aperture diaphragm for regular brightfield work, and/or several light annuli. The light annuli, together with phase objectives, enable the microscopist to make quick comparisons between phase contrast and DIC images. A rotatable polarizer is fitted below the prisms. Use of full objective aperture enables focusing on a thin plane section of a thick specimen without confusing images from above or below the plane that is being focused on; this is called Optical Sectioning. Larger apertures also yield better resolution in optical microscopy.

The color and/or light intensity effects shown in the image are related especially to the rate of change in refractive index, thickness or both in details or adjacent areas of the specimen. The image appears 3-dimensional. This appearance does not represent the true geometric nature of the specimen, but is an exaggeration based on optical thickness, and is not suitable for accurate measurement of actual heights and depths. Optical thickness refers to changes in the light path resulting from change in refractive index or actual thickness or some combination of both of these variables.

As mentioned above, at the gray setting of the movable upper prism, the 3-dimensionality is most marked. Orientation of the specimen can significantly improve the relief-like appearance. Sometimes the rotation of the specimen 180 degrees changes a hill into a valley or vice versa; hence the interpretation of the image must be done with caution. The darker appearance on one side and the lighter appearance on the other side of a detail greatly improve the visibility by giving a pseudo-relief effect.

To configure a microscope for DIC contrast enhancement, the following steps should be considered:

- Place the DIC condenser into the substage condenser holder of the microscope and fit the DIC Wollaston prism into a slotted nosepiece or in slots behind each objective. Using a 10X objective and the condenser at the brightfield (0) position and polarizer in the light path, set up Köhler illumination with a specimen in place on the stage. Move the specimen out of the light path and remove one of the eyepieces.

- Insert a phase focusing telescope into the eyepiece tube and, while looking at the back focal plane of the objective, rotate the screw of the upper prism until you see a diagonal blackish line appearing at the center of the back of the objective. Now slightly rotate the substage polarizer to make the black line appear as black as possible. This, in effect, is adjusting the polarizer so that it is crossed (at a 90 degree angle) with the analyzer that is situated above the upper prism. Make sure the condenser aperture diaphragm is open to 2/3 to 4/5 of the back lens diameter of the objective.

- Remove the focusing telescope and return the regular eyepiece to the eyetube. Rotate the condenser turret to bring the appropriate lower prism into the light path; this is usually marked by the red or white 10 setting on the turret. Move the specimen back into the light path. Now you may use the knob of the upper prism to translate it back and forth laterally to achieve the desired effect or color. You may also rotate the stage to change the orientation of the specimen to improve the effect.

- Similar steps are taken for each objective being used, making sure the microscope is properly configured for Köhler illumination for each objective in turn by adjusting both the field and aperture diaphragms.

These are numerous advantages in DIC microscopy as compared particularly to phase microscopy:

It is possible to make fuller use of the numerical aperture of the system because, unlike phase contrast microscopy, there is no substage annulus to restrict the aperture; Köhler illumination is properly utilized.

There are no confusing halos as may be encountered in phase images.

Images can be seen in striking color (optical staining) and in 3-dimensional shadowed-like appearance. The visibility of outlines and details is greatly improved, and the photomicrography of these images is striking in color and detail.

Regular planachromats or achromats (also suitable for ordinary brightfield work) can be used if the manufacturer states that such objectives are designed for their apparatus.

There are several disadvantages or limitations in DIC:

The equipment for DIC is quite expensive because of the many prisms that are required.

Birefringent specimens such as those found in many kinds of crystals may not be suitable because of their effect upon polarized light. Similarly, specimen carriers, such as culture vessels, Petri dishes, etc., made of plastic may not be suitable. For such specimens, Hoffman modulation contrast may be a better choice.

For very thin or scattered specimens, better images may be achieved using phase contrast methods.

Apochromatic objectives may not be suitable because such objectives themselves may significantly affect polarized light.

Once again, like so many of the contrasting enhancing techniques, we find that manipulation of light at the front focal plane of the condenser and at the rear focal plane of the objective has significant effect upon the appearance of the image that is visualized through the eyepiece.

Contributing Authors

Mortimer Abramowitz - Olympus America, Inc., Two Corporate Center Drive., Melville, New York, 11747.

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

Differential Interference 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 (see Figure 1) 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.

Presented in Figure 1 is the typical optical train configuration for a transmitted differential interference contrast microscope. Semi-coherent wavefronts emitted from localized neighborhoods in the lamp filament pass through a linear polarizer positioned between the light port in the microscope base and the condenser assembly. A compound Nomarski or Wollaston prism is located in or near the condenser aperture (front focal plane), and serves to align and shear incident polarized wavefronts into two orthogonal components. The perpendicular, sheared wavefronts are focused by the condenser lens system into parallel bundles that traverse the specimen plane and respond to refractive index and thickness gradients by deformation according to optical path length parameters of the specimen.

Light waves gathered by the objective converge at the rear focal plane where a second Nomarski prism is located. The objective Nomarski prism recombines sheared and deformed wavefronts into linear and elliptically polarized light that subsequently passes a component through the analyzer (a second polarizer oriented perpendicular to the substage polarizer) located in an intermediate tube above the objective. Finally, the linear polarized light components that emerge from the analyzer recombine through constructive and destructive interference at the image plane in the eyepiece diaphragm (or camera projection lens).

Polarizing Elements for DIC Microscopy

The polarizers employed in DIC microscopy are similar or identical to those used for polarized light observations, but many manufacturers offer matched DIC polarizing filters that have both low and high light transmission efficiencies. At the upper end of the magnification range (40x, 60x, and 100x), polarizers with high transmission efficiencies are preferred for DIC because specimen details are more clearly delineated and larger bias retardation values can be utilized for video and digital imaging. The low transmission polarizing filters are useful for DIC observation using the 10x and 20x objectives, but severely restrict light transmission at higher magnifications.

On most microscopes, the polarizer is mounted directly onto the light port in the base of the instrument or in a filter holder attached underneath the condenser. Polarizers designed to span the light port generally are mounted in a rotating assembly that enables the microscopist to rotate the filter through a 90 to 180-degree angle (see Figure 2(a)). Once the rotating polarizer is positioned at the desired transmission azimuth, it can be secured into place with a locking screw. By convention, the polarizer is oriented with the vibration transmission axis positioned in an East-West direction (left to right when standing in front of the microscope). Microscopes that have the polarizer attached to the substage condenser mounting bracket are provided with a housing that may or may not enable rotation of the polarizer (Figures 2(b) and 2(c)). Polarizers having circular geometry fit into a bracket in the stage and can rotate in fixed increments of 45 degrees with a détente. Other polarizers are mounted in rectangular frames that slide into a slot in the condenser bracket (Figures 2(b) and 2(c)), and often contain thumbwheels that enable rotation through 180 to 360 degrees.

Analyzers are placed between the objective Nomarski prism and the eyepiece observation tubes in microscopes equipped for DIC observation, similar to the location of these components in polarized light microscopes. The analyzer is also a linear polarizer that is positioned with the transmission azimuth oriented at a 90-degree angle with respect to that of the polarizer. By convention, the vibration direction for the analyzer is North-South, which coincides with the East-West orientation prescribed for the polarizer.

The microscope mounting configuration for analyzers is equally as varied as it is for polarizers, and these components are commonly inserted into the optical train throughout a number of locations. In some microscopes, the analyzer is fixed into a rectangular frame (Figure 2(e)) and inserted into a slot in the nosepiece, intermediate tube, or vertical illuminator. Other analyzer designs feature the same frame style, but enable rotation of the analyzer element with a thumbwheel that is often graduated in 10, 45, or 90-degree increments (Figure 2(f)). Polarized light microscopes equipped for DIC observation often house the analyzer in an intermediate tube (see Figure 2(d)) located between the objective nosepiece and the observation tubes. These units are often designed for precision measurements in polarized light and feature 360-degree graduated vernier scales wrapped around the circumference, with a locking mechanism to secure the analyzer in the desired transmission azimuth. In addition, the analyzer is usually mounted on a slider so that it can be conveniently removed from the light path for linearly polarized or brightfield observation. Intermediate tubes for polarized light microscopy also contain a 20 x 6 millimeter DIN standard slot for a quarter-wavelength, full-wave, or de Sénarmont compensator (Figure 2(d) and Figure 5).

Modern microscopes position the polarizer and analyzer in strategic locations with respect to the field lens, condenser, objective and observation tubes. In older microscopes, these polarizing elements can be found installed in a wide variety of locations. It should be noted, however, that placing polarizing elements in or very near a conjugate image plane (the field diaphragm, specimen stage, or eyepiece fixed aperture) is not a good idea, because scratches, imperfections, dirt, and debris on the glass surface can be imaged along with the specimen.

Condenser and Objective Prisms

DIC condenser prisms, which act as beamsplitters to produce an angular shear to incoming polarized light wavefronts, are often mounted in a revolving turret condenser designed to house at least three individual prisms, as illustrated in Figure 3. Turret specifications and configurations vary according to the manufacturer, but they generally contain slots for four to eight auxiliary components, including Wollaston or Nomarski prisms, phase contrast annular rings, or darkfield light stops. The condenser turret illustrated in Figure 3 contains seven openings, three of which are filled with phase contrast annuli and three with DIC prisms. The open slot is used for brightfield observation.

Each condenser DIC prism (also termed compensators or auxiliary prisms) must be specifically matched to a narrow range of objective numerical apertures, so a particular prism may only work for one or two objectives (for example, the 10x and 20x). As a result, between three and five condenser prisms must be employed to match the entire objective magnification range between 10x and 100x. Some manufacturers tailor each condenser prism for a particular objective, thus requiring up to seven condenser prisms to span the entire spectrum of dry and oil immersion objectives having varied numerical apertures.

Condenser DIC prism inserts are constructed with anodized circular aluminum plates having the combination prism (usually circular in shape) secured in a precise orientation with optical cement. DIC prism wedges are very thin and cut with close tolerances to ensure that angular shear values match those required by the objective numerical aperture. The polished plates must be handled carefully to avoid contamination from fingerprints, oil, dirt, and debris. Each prism frame contains a slot or pin that mates to a corresponding partner in the condenser turret in order to define and secure alignment of the condenser prism with respect to the objective prism and the polarizer (and analyzer) axes.

Positioned between the objective and the analyzer is a second Nomarski compound prism that acts to recombine wavefronts sheared by the condenser prism (Figures 1, 4, and 5). This prism, often termed the objective or principal DIC prism, can either be matched to a specific condenser prism, or a single objective prism can serve to recombine wavefronts having the spectrum of shear angles represented by all of the condenser prisms. A majority of the microscope manufacturers configure their DIC microscopes to employ a single objective Nomarski prism, which is mounted in a rectangular frame that slides into the nosepiece (see Figure 4). In order to introduce bias retardation into the DIC optical system, the objective prism rides on a gliding support that can be translated back and forth across the microscope optical axis by means of a micrometer control knob.

Objective DIC prisms designed to introduce bias retardation through translation across the microscope optical axis are fabricated in a rectangular shape with the long axis corresponding to direction of prism shear (Figure 4(b)). In a properly aligned DIC microscope, the condenser prism is imaged onto the objective prism by the combined action of the condenser and objective lens systems. As a result, the wavefront shear produced by the condenser prism is exactly matched at every point along the surface of both prisms (which are inverted with respect to one another). Translating either prism along the shear axis produces a wavefront mismatch (bias retardation), which, in turn, introduces an optical path difference that is uniform across the microscope aperture. Translating the objective prism with the micrometer control is the most popular mechanism for producing bias retardation in DIC microscopes.

Several objective prism slider designs from various manufacturers are illustrated in Figure 4. All of the compound prisms are housed in rectangular frames with a translational control knob positioned at the end of the frame. The control knob is employed to shift the prism position laterally along the shear axis (Figure 4(b)) in order to introduce bias retardation (or a net wavefront optical path difference), into the differential interference contrast optical train. Nomarski prism sliders designed for reflected light DIC microscopy also contain a second control knob (Figure 4(c) and 4(d)) that enables adjustment of the prism height to match varying rear focal plane positions throughout the entire objective magnification and numerical aperture range. When the microscope is operated in another imaging mode, the objective prism frame can be conveniently removed from the optical path by sliding the entire frame without removing it from the nosepiece or intermediate tube housing.

In DIC microscopes designed to introduce bias retardation using a de Sénarmont compensator, the objective Nomarski prisms are secured with fixed mounts that slide into the nosepiece above the objectives (Figure 5(c)). Microscopes using de Sénarmont compensators require a separate objective prism for each objective, but can often utilize the same condenser Nomarski prism for two or more objectives. The fixed objective prism mounts (Figure 5(c)) are easily removed from the light path by sliding the frame away from the microscope nosepiece.

A typical polarizer and quarter-wavelength retardation plate configuration for de Sénarmont differential interference contrast is illustrated in Figure 5(a). This unit is fitted over the field diaphragm adjustment knob on the microscope base and, after alignment of the retardation plate optical axis and polarizer transmission azimuth, secured to the knob with a locking setscrew. The polarizer axis is marked on the front of the de Sénarmont compensator unit, and graduated rulings enable the operator to qualitatively determine the approximate amount of bias retardation introduced into the system when the polarizer is rotated. The locking knob can be utilized to hold the polarizer immobile with respect to the quarter-wavelength plate. For brightfield observation or enhanced contrast techniques without DIC, the entire polarizer and retardation plate assembly can be removed from the optical path by swinging out the hinged upper section.

An alternative technique for de Sénarmont DIC compensation is to place the quarter-wavelength compensator plate in the optical train between the objective Nomarski prism and the analyzer. In this case, either a standard linear polarizer is employed beneath the condenser prisms or the de Sénarmont compensator illustrated in Figure 5(a) is set with the polarizer axis parallel to the retardation plate fast axis (only linear polarized light emerges from the compensator) and perpendicular to the analyzer. An intermediate tube designed for introducing de Sénarmont DIC compensation after the objective prism is illustrated in Figure 5(b). In this case, bias retardation is introduced into the optical system by rotating the analyzer, rather than the polarizer.

Equipped with a Bertrand lens for observing the objective rear focal plane, the intermediate tube presented in Figure 5(b) also has a DIN standard slot able to accommodate a variety of retardation plates, including a de Sénarmont compensator (shown in the Figure). The analyzer can be rotated by a precision graduated vernier mechanism, which enables the operator to quantitatively determine the level of bias retardation introduced into the DIC optical system. With de Sénarmont compensators, bias retardation values ranging between one-twentieth and a full wavelength can be easily measured with an accuracy of 0.15 nanometers. In addition, the Bertrand lens can be conveniently inserted into the light pathway by means of a thumbwheel in order to observe events occurring at the objective rear focal plane. The use of a Bertrand lens or phase telescope to monitor the objective focal plane is critical to DIC microscope alignment, as discussed below.

In DIC microscopy, contrast is a function of specimen orientation, and the wide variety of geometries encountered often requires repositioning of the specimen to maximize contrast effects for observation of the target structures. Use of standard rectangular mechanical stages to observe DIC specimen is hampered by the limited range of rotational motion exhibited by these components, although the x-y translation mechanism eases the burden of carefully examining the entire contents of a microscope slide. In order to allow specimens to be easily rotated and manipulated to present the optimum azimuthal contrast, a circular rotation stage similar to that illustrated in Figure 6(a) is recommended for DIC microscopy. The circular stage enables 360-degree rotation of the specimen, which is often necessary with extended linear specimens (such as diatoms or filamentous structures) that display extreme azimuthal contrast effects in DIC. Translation of the specimen during observation is aided by the utilization of a graduated mechanical stage accessory (Figure 6(b)) that attaches directly to the circular stage and securely clamps a microscope slide. The stage should be centered along the microscope optical axis, either by adjusting the position of the stage itself or by centering the objectives and condenser with respect to the stage.

Objectives designed for differential interference contrast must be free of strain or birefringent occlusions that depolarize light and lead to image degradation. In the past, microscopists were limited to strain-free achromat and fluorite objectives intended for observations in polarized light, but modern objectives having higher correction factors can now be employed for DIC microscopy. Termed universal objectives, these advanced lens systems can often be used for combined DIC, fluorescence, phase contrast, and brightfield microscopy without having to change objectives. In addition, newer apochromatic objectives have been designed that are sufficiently strain free to enable their use in polarized light and differential interference contrast observation, dramatically improving the image quality and resolution at higher magnifications.

Differential Interference Contrast Microscope Alignment

Before attempting to configure a microscope for observation in differential interference contrast, inspect the instrument to ascertain whether all of the necessary components are present, and free of lint, dust, and debris. Objective and condenser lens elements that contain stress signatures can degrade images in DIC, as can dirty lens surfaces, scratches, and contaminating foreign material in the optical pathway. Proper alignment of the microscope is essential to achieving optimum results and producing images that display pseudo three-dimensional and shadow-cast effects. Many of the steps outlined in the following procedure are only necessary when first aligning the microscope for DIC and do not require repetition for daily observations. Other steps should be taken each time the microscope is used for DIC investigations.

Preliminary Microscope Inspection - Examine the microscope carefully to ensure that all necessary DIC components are installed, or available and ready for use when necessary. Remove the condenser, disassemble the turret, and inspect the condition of the Nomarski or Wollaston prisms. The surfaces of these compound prisms should be clean and free of dust and debris. Because they are housed within the condenser turret, DIC condenser prisms rarely become contaminated with fingerprints, but dust and lint can easily flow into the turret and land on one of the flat quartz surfaces. To clean a contaminated prism surface, use a rubber balloon to remove loose fibers and dust, and/or gently wipe the surface with lens tissue or moist soft cotton. Be careful not to scratch the surfaces. The same treatment should be afforded to the objective prism(s), condenser and objective external lens elements, microscope eyepiece lenses, and the field lens at the field diaphragm port in the base of the microscope (or attached to the pillar of an inverted microscope). After ensuring the critical components are clean, reassemble the microscope, install the polarizer and analyzer, and then align the optical system for Köhler illumination.

Install the Polarizer and Analyzer - With the microscope disassembled (condenser, DIC prisms, and at least one objective removed), install the polarizer and analyzer in their positions beneath the condenser and above the objective, respectively. In a manner similar to polarized light microscopy, the polarizer and analyzer are positioned so their transmission azimuths are crossed at a 90-degree angle (perpendicular) to one another. The polarizer, which is mounted between the light source and the condenser, is traditionally oriented in an East-West direction, or left to right when facing the microscope. In some cases, the positions of both the polarizer and analyzer are pre-determined by their fixed position in the mounting frames, and these components can only be inserted into the microscope optical pathway with a single orientation. Usually, a marker on the polarizer mount indicates the transmission direction, but some microscopes are equipped with a rotating polarizer mount that is graduated in degrees (Figure 2). The analyzer may also be rotatable with a graduated knob, and/or may contain a mark indicating the transmission axis.

When the polarizer and analyzer are crossed (transmission axes oriented at a 90-degree angle), the viewfield appears very dark when observed through the eyepieces. This condition is referred to as maximum extinction. If a significant amount of light passes through the microscope and the viewfield is not dark (or almost black), check to ensure the polarizer and analyzer are crossed. After crossing the polarizers, insert the condenser and objective, but do not install the objective Nomarski prism slider (or fixed mount). Rotate the condenser turret to the brightfield position (the slot lacking a phase plate or DIC prism). The viewfield should remain dark, but if either of these components (the condenser or objective) contains strained lens elements, some light may pass through. Remove one of the polarizing elements from the optical train (either the polarizer or analyzer) before proceeding to the next step.

Establish Köhler Illumination - Before proceeding with DIC configuration (after the polarizers are installed), the microscope optical system should be aligned for brightfield specimen observation using the standard Köhler technique. When properly configured, an image of the light source (usually a tungsten-halogen lamp) should be projected onto the condenser aperture diaphragm plane by the collector lens housed in the lamphouse or along the optical train inside the microscope frame base. Simultaneously, the condenser lens system also projects an image of the field diaphragm into the specimen conjugate plane (at the microscope stage). After the lamp filament has been centered (most modern lamphouses contain a pre-centered lamp), close the condenser aperture diaphragm and align the condenser with the microscope optical axis in brightfield illumination (the condenser turret is set to the 0 or B position). Bring the diaphragm into focus, superimposed on a focused specimen using the 10x objective, and open the iris leaves until only a small portion of the diaphragm is visible at the peripheral edges of the viewfield. Similar steps are taken for each objective being used, making sure the microscope is properly configured for Köhler illumination for each objective in turn by adjusting both the field and aperture diaphragms. During the course of daily observations in DIC, the microscope should be periodically checked to ensure that Köhler illumination is maintained.

Inspect the Objective Rear Aperture - After configuring the microscope for Köhler illumination, insert the polarizer and analyzer and examine the objective rear focal plane with a phase telescope or Bertrand lens (conoscopic observation mode). If the polarizer and analyzer are properly positioned and the microscope perfectly aligned, a dark extinction cross will appear in the objective aperture, as illustrated in Figure 7(a). The arms of the extinction cross should be oriented vertically and horizontally, with a small amount of light appearing at the four corners of the aperture (Figure 7(a)). Bright spots in the cross or highly birefringent regions, which affect the integrity of the extinction cross, are an indicator of strained optics. In addition, dust and lint particles positioned near a conjugate aperture focal plane (condenser or objective) will appear bright when viewed at the objective rear aperture. If birefringent spots are present, check another strain-free objective to determine whether the first objective or the condenser lens system is strained. Remove any contaminating dust from the objective or condenser lens surfaces and replace strained optical components (if possible) before proceeding to the next step.

Objective DIC Prism Alignment - Install the objective prism by either inserting the slider (Figure 4) or a prism confined to a fixed mount (for systems utilizing de Sénarmont bias retardation; see Figure 5). Once the prism is in position, examine the objective rear focal plane once again with the phase telescope or Bertrand lens. The viewfield should now appear very bright, but featureless, with a single dark interference fringe extending across the diameter of the aperture at a 45-degree angle (see Figure 7(b)) along the shear axis. Depending upon whether the microscope is upright or inverted, the interference fringe will traverse the objective rear aperture in a northeast-southwest (upright) or northwest-southeast (inverted) direction. In either case, the interference fringe should be well defined, as illustrated in Figure 7(b), and positioned in the center of the aperture.

In some DIC microscope designs, the objective prism is fixed (de Sénarmont compensation), while in others the prism can be translated back and forth across the optical axis by means of a positioning screw mechanism in the slider frame. In the latter case, slowly twist the adjustment knob while observing the objective rear focal plane through the telescope or Bertrand lens. As the knob is turned, the interference fringe should move away from its central position to either the upper or lower half of the bright rear aperture. Alternatively, turning the polarizer in a de Sénarmont compensator will produce the same effect.

Condenser DIC Prism Alignment - Remove the objective prism from the optical train, and swing the lowest aperture condenser prism (for use with the 10x objective) into position by rotating the condenser turret. The appropriate position is usually marked by the red or white 10 setting on the turret (or a similar code, such as L). Refocus the phase telescope or Bertrand lens to observe the interference fringe that appears in the objective rear focal plane. Once again, a single fringe should be present having the same orientation as the fringe produced by the objective prism (northeast-southwest for upright microscopes or northwest-southeast for inverted microscopes). The interference fringes for the condenser and objective prisms should appear almost identical and should have the same orientation along the shear axis.

In order to clearly observe the condenser prism interference fringe using high-numerical aperture condensers designed for oil immersion, it may be necessary to remove the front lens assembly using the swing-lens control lever. If the fringe appearing in the objective aperture is not correctly positioned, it may be necessary to adjust the orientation or alignment of the condenser prism. In most cases, condenser prisms are assembled in a protective circular aluminum frame (Figure 3) with a notch or pin (or a lock-down screw) to ensure correct positioning within the condenser turret. Occasionally, a condenser prism can be forced into the turret without proper alignment, which will be apparent when the interference fringe is examined. If a condenser prism appears to be out of alignment, check with the microscope manufacturer for instructions on proper adjustment of the condenser DIC prisms.

Specimen Observation - With the microscope aligned for Köhler illumination, the polarizer and analyzer crossed, and both prisms (objective and condenser) installed, place a thin transparent mounted specimen (such as a buccal mucosa epithelial cell preparation) on the stage. Adjust the microscope for maximum extinction, and focus the specimen while observing the procedure through the eyepieces in orthoscopic mode (no Bertrand lens or phase telescope). The image observed in the viewfield should appear very dark gray, almost black, at maximum extinction with bright highlights in regions having sharply defined thickness or refractive index gradients (for example, the cellular membrane and nucleus; see Figure 8(b)). Spherical specimens with a high refractive index, such as immersion oil droplets, may even act as tiny lenses, and appear with a sharply defined interference fringe or band traversing the central region oriented in the same direction as the fringes are when observed in the objective rear aperture.

While observing the focused specimen image, translate the objective DIC prism using the slider knob or rotate the polarizer (or analyzer) in a microscope equipped for de Sénarmont compensation. This action is termed introduction of bias retardation, and will translate the interference fringe bisecting the specimen along the shear axis and produce a corresponding change to specimen appearance. Shifting the prism in one direction (positive bias) will lighten specimen features at one edge while darkening the same features on the opposite edge and simultaneously lighten the background (see Figure 8(a)). In general, the specimen assumes a pseudo three-dimensional appearance with a shadow-cast effect oriented in the same direction as the shear axis. Moving the prism to the other side of the microscope optical axis (negative bias) will reverse the light and dark specimen regions (compare Figure 8(a) with Figure 8(c)).

At maximum extinction with all DIC components properly installed and aligned, the objective rear aperture appears dark gray (almost black) and relatively uniform when observed with a phase telescope or Bertrand lens (Figure 7(c)). In most cases, the central region of the rear aperture appears jet black while some evidence of light appears in the four quadrants at the periphery. The extinction cross should generally appear quite similar to that observed with crossed polarizers alone, but usually is much darker and covers a larger region of the objective rear aperture. The bright areas surrounding the periphery (Figure 7(c)) result from an artifact that arises through partial depolarization of light at the surface of the polarizers and lens elements in the condenser and objective.

Differential interference contrast images can be significantly improved by masking the bright regions at the periphery of the extinction cross in the objective rear aperture. This is accomplished by reducing the size of the condenser aperture diaphragm to eliminate the bright edges. In general, the objective rear aperture size should be reduced with the condenser diaphragm to approximately 75 or 80 percent of the full aperture. When the optical system is in perfect alignment, the extinction cross appears upright (see Figure 7) and can be observed to consist of two broad interference fringes, each shaped in a right angle and meeting at the center of the objective rear aperture (the fringes can also be visualized in orthoscopic mode in lower quality microscopes).

On some microscopes, the condenser and objective prism positions can be adjusted to yield a more uniform fringe pattern, resulting in the central region of the aperture appearing darker and more uniform. This task is accomplished to loosening and rotating (or raising or lowering) the condenser prism or by uncrossing the polarizer and analyzer by a couple of degrees. Occasionally microscopes contain setscrews that allow for adjustment of the condenser and objective prisms, but models so equipped are becoming rare. As a final check on microscope alignment, adjust the condenser focus knob while examining the extinction pattern in the objective rear aperture to determine whether it can be improved. Note that a significant repositioning of the condenser may degrade optical performance by separating the overlap between conjugate interference planes of the objective and condenser DIC prisms.

Adjusting the bias retardation by translating the objective prism, or rotating the polarizer in a de Sénarmont configuration, dramatically improves the image appearance (over that observed at maximum extinction) and increases contrast. This maneuver is essential for imaging specimens in differential interference contrast, and represents the last step in the adjustment of the microscope optical train. In many cases, a gradient of light appears across the entire field of view when observing DIC images. This occurs in addition to the presence of light and dark intensities at opposite edges of the specimen, and is due to a broad and indistinct field fringe artifact produced by the optical system. Microscopes having well-matched optical components maximize the size of the field fringe, which can become so broad and evenly distributed that the entire field appears a uniform medium gray color. In most cases, however, some evidence of the fringe remains, and the viewfield exhibits a shallow gradient of light intensity (medium to light or darker shades of gray) from one peripheral edge to the other. This artifact is inherent in a particular optical configuration and should be ignored when observing and collecting images of DIC specimens.

Compensators in DIC Microscopy

Specimen contrast can also be increased by introducing a retardation plate (or compensator) into the optical pathway in a DIC microscope. In general, a full-wave (also termed a first-order compensator) plate is inserted in an intermediate tube between the objective prism and the analyzer, although the plate can also be situated after the polarizer but before the condenser prism. These plates exhibit a retardation level of an entire wavelength at a specified value in the green region (usually near 550 nanometers), and result in the specimen displaying yellow and blue Newtonian interference colors along refractive index and thickness gradients. The background is rendered magenta due to the subtraction of green wavelengths from white light.

In a de Sénarmont or a standard (translatable) Nomarski prism DIC microscope configuration, when the objective prism is positioned with the extinction interference fringe in the center of the optical pathway, a pattern similar to that displayed in Figure 7(b) is observed at the objective rear focal plane (provided the condenser prism is removed from the optical path). If a full-wave retardation plate is then placed between the objective prism and the analyzer, the interference pattern illustrated in Figure 9(a), which displays a spectrum of Newtonian interference colors, appears in the objective rear focal plane. Removing the objective prism from the light path and inserting a condenser prism yields the pattern presented in Figure 9(c). When both the objective and condenser prisms are present in the optical train and adjusted to the extinction position, a magenta color is visible in the objective rear focal plane (Figure 9(b)).

Translating the objective Nomarski prism or rotating the polarizer in a de Sénarmont compensator configuration will shift the Newtonian interference pattern color illustrated in Figure 9(b). Introducing negative bias will shift the Newtonian colors to subtractive values (yellow), while shifting the prism to positive bias values will result in additive colors (blue). The colors produced by specimen gradients can be compared to a Michel-Levy reference chart in order to determine the magnitude of optical path differences.

DIC Microscope Configurational Errors

When a differential interference microscope is properly configured, the resulting images display a realism manifested in pseudo three-dimensionality through a shadow-cast effect that appears to originate from a highly oblique angle (see Figure 10(a)). However, even slight alignment errors can result in a deterioration of minute specimen detail, and serious mistakes in configuration can yield a dramatic loss of contrast that renders the image useless. The most common errors in DIC microscope configuration result from polarizers that are not crossed, a mismatch between the objective and condenser prisms, a missing condenser DIC prism, or incorrect setting of the condenser iris aperture. Other problems can arise, especially if the condenser and/or objectives are not strain free, but these mistakes are rare if the microscope is designed specifically for imaging specimens in DIC illumination.

Ensuring that the polarizers are crossed and positioned in the correct orientation was discussed previously in the section on microscope alignment. Occasionally, either the polarizer or analyzer can become accidentally rotated out of position by brushing against the thumbwheel or careless use of the instrument. As a result, overall specimen contrast is reduced, but many features often remain visible. This type of mistake can be eliminated by utilization of a fixed polarizer and analyzer or by ensuring that the locking knob is set to suspend polarizer rotation. Most polarizers housed in rotating mounts contain marks that indicate the position of the transmission azimuth. A careful check of the polarizer and analyzer orientation prior to commencing DIC observations will avoid errors with these components.

Accidental mismatch between the objective and condenser prisms is another common source of errors in differential interference contrast microscopy. Because of the shear angle discrepancies between prisms not designed to work together, the resulting image exhibits extremely poor contrast (see Figure 10(c), which cannot be corrected by translating the objective prism or rotating the polarizer in a de Sénarmont compensation microscope. The condenser turret is usually marked with a code that identifies the prism placed into the optical pathway. Inspection of the exterior code order to ensure that condenser prisms are positioned in the correct slot will help to avoid this type of error. Also, the prism code inscribed on the objective barrel should match the code for the condenser prism in use. In general, prism mismatches occur when the magnification is changed, but the condenser turret is not rotated to insert a matching prism into the optical pathway. Occasionally, a condenser prism may be missing from the condenser or the condenser turret rotated into a position that does contain a prism (for example, the brightfield position; see Figure 3). The result is an image similar to that illustrated in Figure 10(c), which lacks contrast and does not display the high resolution obtainable with differential interference contrast.

The condenser iris diaphragm is typically employed to adjust depth of field and contrast for images observed in the eyepieces or recorded digitally and on film. In differential interference contrast microscopy, wide aperture sizes are useful for optical sectioning experiments, where a shallow depth of field is required, but high resolution is also necessary. Often, the compromise between maximum contrast afforded by using smaller apertures is offset by the elimination of interfering specimen features located away from the plane of focus. If the condenser aperture is not opened sufficiently (to about three-quarters to four-fifths of the objective aperture), diffraction artifacts can obscure important specimen detail and seriously degrade image appearance (Figure 10(b)). In general, diffraction effects can be avoided by maintaining the condenser iris diaphragm size between 80 to 90 percent of the objective aperture.

Additional errors can occur due to spherical aberration when using thick specimens immersed in water, or when the glass microscope slide, culture dish, or coverslip is too thick. These mistakes are manifested by images that are not sharply focused and lack the shadowed relief that is characteristic of DIC images. Correction collars on high magnification dry objectives can often be adjusted to correct for spherical aberration, but specimens that are too thick must be replaced with thinner sections. When examining cells in culture, do not use DIC optical systems with culture vessels made from injection-molded polymers. These materials display stress birefringence and will produce confusing images. Instead, use glass vessels designed especially for DIC microscopy, which are available from several manufacturers. In general, careful attention should be paid to selection of high-quality glass microscope slides and coverslips manufactured to precise tolerances, and the glass should be thoroughly cleaned and dried prior to preparation of specimens.

Illumination Sources for DIC Microscopy

At lower magnifications (10x through 40x), a 50-watt quartz halogen lamp can provide enough light for satisfactory observation and recording of images in DIC. However, at the highest magnifications (60x and 100x), at least a 100-watt tungsten-halogen light source is recommended. The illumination intensity required by the light source is dependent upon the transmission percentage of the polarizer and analyzer, and many older DIC microscopes are equipped with polarizing elements that have low transmission values (20 percent or less). However, later model microscopes are often equipped with high transmission polarizers (greater than 30 percent), which enable a higher flux density to pass through the optical system, providing adequate light for observation and imaging at the highest magnifications.

Although coarse structural detail, such as the edges of cells and larger intracellular components (nuclei) can be easily visualized with a high-intensity tungsten-halide lamp, in order to observe the finest structural details, very intense illumination having a narrow bandwidth is essential. Mercury arc-discharge lamps feature a high-energy peak in the central portion of the visible spectrum at 546 nanometers (green), which can be further refined by passage through a narrow bandwidth (10 nanometer) interference filter centered at 546 nanometers. In addition, a light scrambler can be employed to enhance the narrow bandwidth illumination to achieve the highest possible resolution. Note that the objective aperture should be fully illuminated, regardless of the light source, for the best results.

Conclusions

The correct placement and orientation of optical components in a differential interference contrast microscope is critical to optimum performance, regardless of the target magnification and resolution. Improper adjustment of even a single component can result in serious image degradation and upset the shadow-cast effect and resolution of the instrument. The two compound prisms and their associated polarizers in DIC microscopy are mirror image pairs. Light waves passing through the initial polarizer and condenser prism are imaged onto the objective prism and analyzer to produce the final image. All four of these components must be in the proper orientation in order for DIC to function properly. Contrast is generated either by altering the lateral position of the objective prism, or by rotating the polarizer in de Sénarmont compensation instruments. In this manner, the optical path difference between wavefronts is increased or decreased to produce contrast in the specimen.

It should be noted that contrast in DIC is a function of gradients in the specimen optical path length, which are rendered in pseudo three-dimensional relief when the microscope is properly adjusted. Because contrast arises from a combination of refractive index fluctuations and/or thickness variations, apparent gradients observed with the technique can correspond to real changes in specimen topography or may be a function, for example, of localized protein concentration gradients. Only independent knowledge of the structural properties relating to the specimen can help determine the absolute nature of contrast effects in DIC microscopy.

In addition, even when it is determined that shadow-cast effects can truly be ascribed to height changes in the specimen, there is no inherent mechanism in DIC to indicate which features represent plateaus or valleys at a particular setting of the objective prism. A qualitative solution to the problem is to locate a known specimen characteristic, such as a scratch in the glass, and observe how the feature responds to bias retardation as the objective prism is translated across the optical axis. Integral specimen features having a similar response will be either valleys or plateaus, while those with opposite responses will have a reverse orientation.

When examining unknown specimens with differential interference contrast, the microscopist should be alert to interference colors arising at the edges, which are normally featureless and exhibit either a very light or dark color, depending upon orientation. The presence of interference colors indicates that the specimen may be birefringent and, consequently, not a good candidate for DIC observation. This fact can be confirmed by examining the specimen under crossed polarized illumination with both the objective and condenser DIC prisms removed from the optical pathway.

Among the limitations imposed on differential interference contrast microscopy are the expensive birefringent Nomarski or Wollaston prisms necessary to perform the technique. These components are far more expensive than those required for phase contrast or Hoffman modulation contrast microscopy, which may serve as alternative techniques, especially when observing living cells in plastic vessels. In addition, very thin or scattered specimens often produce better images having more contrast when phase contrast is used instead of DIC. Older apochromatic objectives may not be suitable for DIC observation because the objectives themselves can significantly affect polarized light. The microscopist should check with the manufacturer before purchasing high-quality apochromatic objectives in conjunction with DIC optical components.

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., 110 Commerce Drive, Allendale, New Jersey, 07401.

Edward D. Salmon - Department of Cell Biology, The University of North Carolina, Chapel Hill, North Carolina 27599.

Mortimer Abramowitz - Olympus America, Inc., Two Corporate Center Drive., Melville, New York, 11747.

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

Differential Interference Contrast

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. Because phase differences are undetectable to the human eye, and are not readily observed in a microscope under brightfield illumination, the light path through the microscope must be suitably modified in order to produce satisfactory images of phase specimens. Many popular contrast-enhancing techniques rely on the ability of phase specimens to modify the optical path difference between waves traversing the biological material and those passing through the surrounding medium.

Perhaps the most fundamental distinction between differential interference contrast and phase contrast microscopy is the optical basis upon which images are formed by the complementary techniques. 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. Alternatively, specimen features that have relatively low thickness, or a refractive index less than the surrounding medium, are rendered much lighter when superimposed on the medium gray 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. Steep gradients in path length generate excellent contrast, and images display a pseudo three-dimensional relief shading that is characteristic of the DIC technique. Regions having very shallow optical path slopes, such as those observed in extended, flat specimens, produce insignificant contrast and often appear in the image at the same intensity level as the background.

Aside from the differences in contrast-formation mechanisms, DIC and phase contrast images differ in a number of other features. Illustrated in Figure 1 are several digital images comparing specimens captured in DIC and phase contrast. A human buccal mucosa epithelial (cheek) cell, revealing the nucleus, cytoplasmic inclusions, and numerous bacteria on the upper surface, is presented in Figure 1(a), imaged with differential interference contrast. The same viewfield with phase contrast illumination is illustrated in Figure 1(b). The phase contrast image features pronounced halos around the cellular periphery and nucleus, which are absent in the differential interference contrast image. Optical sectioning DIC microscopy investigations (not illustrated) reveal that the bacteria are present (almost exclusively) on the membrane surface that is bathed in surrounding media as opposed to lying on the underside of the cell. This fact cannot be unambiguously determined with phase contrast.

In Figure 1(c), a thick section of murine kidney tissue imaged with differential interference contrast reveals a bundle of cells enclosed within a tubule. A phase contrast image (Figure 1(d)) of the same area is confusing and disturbed by the presence of phase halos outside the plane of focus. However, several of the cellular nuclei appear visible in the phase contrast image, which are not distinguishable in DIC. Relatively high magnification views of an Obelia polypoid annulated stem perisarc in DIC and phase contrast are illustrated in Figures 1(e) and 1(f), respectively. In DIC, (Figure 1(e)) the annular structure appears hemispherical with internal rays that emanate radially from the stem. In addition, granular particles are visible within the stem structure, but anatomical detail is largely undefined. The phase contrast image (Figure 1(f)) is confused by halos around the annular rings and within the stem.

A primary advantage of differential interference contrast over phase contrast is the ability to utilize the instrument at full numerical aperture without the masking effects of phase plates or condenser annuli, which severely restrict the size of condenser and objective apertures. The major benefit is improved axial resolution, particularly with respect to the ability of the DIC microscope to produce excellent high-resolution images at large aperture sizes. Illustrated in Figure 2 are digital images acquired with a Bertrand lens of the objective rear aperture in differential interference contrast (Figure 2(a)) and phase contrast (Figure 2(b)) microscopes. In both cases, the objective is a 40x fluorite having a numerical aperture of 0.75, and the condenser aperture diaphragm is opened to the largest diameter size. Even with the polarizer in place and a Nomarski prism installed in the condenser, the DIC aperture is much larger than the phase contrast aperture, primarily because the phase condenser annulus restricts a significant portion of the illumination that would ordinarily traverse the microscope optical train.

Orientation Effects

Phase contrast lacks the pronounced azimuthal effect inherent in differential interference contrast, which is manifested by asymmetrical orientation of the beamsplitting Nomarski (or modified Wollaston) prisms with respect to the microscope optical axis and polarizers. The absence of orientation effect occurs because the annular phase plate in a phase contrast microscope condenser (Figure 2(b)) is rotationally symmetric through 360 degrees and illuminates the specimen evenly from all angles. As a result, the phase contrast image is independent of orientation and not subject to rotational-dependent contrast effects. For many specimens imaged in differential interference contrast, a prerequisite orientation in order to achieve maximum contrast (either parallel or perpendicular to the shear axis) restricts freedom of specimen rotation, especially for linear or closely-spaced periodic structures.

Azimuth contrast effects in differential interference contrast can be utilized to advantage by equipping the microscope with a 360-degree rotating circular specimen stage. Originally designed for observations in polarized light microscopy, circular stages enable the operator to rotate the specimen with respect to the prism shear axis in order to maximize or minimize contrast effects for selected specimen features. Contrast in DIC microscopy achieves a minimum level for linear phase specimens that extend along the direction of shear, but can be varied significantly by rotating the stage by a few degrees. Non-linear specimens, such as cells, tissue sections, particles, and amorphous polymers, do not display a significant azimuth effect in DIC, and can usually be satisfactorily imaged in a variety of orientations.

Presented in Figure 3 are two linear specimens having a significant amount of periodicity and asymmetry imaged in both phase contrast and DIC. In both cases, contrast in the images obtained from DIC is largely dependent upon the orientation of the specimen with respect to the shear axis of the microscope, while the phase contrast image features are independent of specimen rotation around the microscope optical axis. The images in Figures 3(a) and 3(b) were obtained in DIC from a mounted specimen of a bilaterally symmetrical elongated pennate diatom frustule. When the long axis of the diatom is aligned parallel to the Nomarski prism shear axis (Figure 3(a)) ridges and pores in the frustule are readily observed. The white arrow in the upper right-hand corner of Figure 3(a) denotes the Nomarski prism shear axis for the DIC images illustrated in Figure 3. Rotating the diatom by 90 degrees (perpendicular to the shear axis) reduces contrast in the frustule image and obscures important details present in the microstructure. For example, the longitudinal ridges readily observed at the edges and center of the frustule in Figure 3(a) are absent in Figure 3(b). Azimuth effects are absent in the same specimen when imaged using a phase contrast optical system (Figure 3(c)), which displays similar contrast regardless of the specimen orientation. Note that the image contrast in Figure 3(c) more strongly resembles that displayed by the DIC image in Figure 3(b) rather than the high contrast image presented in Figure 3(a). In this case, the benefits of azimuthal effects resulting from specimen orientation in differential interference contrast are clearly evident.

A second example of azimuth effects that are present in DIC, but lacking in phase contrast, is illustrated in Figures 3(d) through 3(f). The specimen is a semi-transparent ctenoid fish scale containing diamond-shaped inclusions that severely lack contrast and are difficult to distinguish when superimposed over the striated body spines of the scale. When the ctenoid scale is oriented with the body spines parallel to the shear axis in a DIC microscope (Figure 3(d)), the transparent rectangular inclusions become visible and mask the body spines. Rotating the microscope stage by 90 degrees reduces the contrast level of the inclusions, and the striated spines become clearly visible (Figure 3(e)). In phase contrast (Figure 3(f)), the inclusions are present with accompanying halos, as well as the striated spines, irrespective of the specimen orientation.

The correlation between image contrast and specimen orientation in differential interference contrast can often be used to advantage in the investigation of extended linear specimens. For example, the highly ordered structures in diatom frustules and similar specimens can overlap, leading to confusing images when observed in phase contrast and similar contrast-enhancing techniques. By capturing images at several orientations, DIC microscopy is often able to present a more clear understanding of the complex morphology present in many extended, linear specimens. In addition, when optical sectioning methodology is coupled to azimuth-specific imaging, differential interference contrast microscopy can often reveal features that are difficult, or impossible, to distinguish using alternative techniques.

Halo and Shade-Off Artifacts

The halo and shade-off artifacts that plague the phase contrast optical system are largely absent in differential interference contrast images. In positive phase contrast, specimen features with a higher refractive index than the surrounding medium are encircled with a bright fringe (halo) that often obscures edge detail. Although halos can be suppressed to a limited degree by configurational modifications to the objective phase plate design, they cannot be eliminated entirely. In some cases, differential interference contrast images suffer from excessive brightness along edges having very steep optical gradients, but this effect usually occurs when bias retardation values are too low, and can be circumvented by proper adjustment of the Nomarski prism (or de Sénarmont compensator polarizer) position.

The presence and severity of halos in phase contrast is dependent, in part, on the refractive index difference between the specimen and the surrounding medium. Often, phase contrast specimens contain a wide variety of structures that display wildly fluctuating refractive indices, which together yield complex images. Areas having a lower refractive index than the surrounding medium exhibit dark halos as opposed to the bright halos that can be observed surrounding higher refractive index features. Halos arise because of the configurational parameters of the phase contrast microscope optical system. A small portion of incident light deviated by the specimen passes through the objective phase plate due to the spherical nature of diffracted wavefronts. Smaller specimen features give rise to larger diffraction angles, but large extended structures can produce diffracted wavefronts having shallower angles that often fall within the objective aperture zone occupied by the phase plates.

A comparison between phase contrast and DIC images with respect to halo artifacts is presented in Figure 4 for three commonly imaged transparent specimens. Human erythrocytes (red blood cells) exhibit a distinctive halo surrounding the disk-shaped cells when imaged in positive phase contrast at medium and high magnifications (Figure 4(a)). Note that the central portion of the erythrocyte disks is lighter than the periphery due to a reduced optical path length in this region. When the same viewfield is examined in differential interference contrast (Figure 4(b)), halo artifacts surrounding the cells are absent, but the images acquire a shadow-cast appearance oriented along the Nomarski prism shear axis (northwest to southeast), which is manifested by the upper left portion of the outer ridges being much lighter in appearance than the corresponding ridges on the opposite side (lower right) of the cells.

Living cells grown in monolayer culture are frequently observed and imaged using both phase contrast and differential interference contrast microscopy. Several adherent HeLa cells in culture on glass coverslips are illustrated in Figure 4(c) with phase contrast optics. Halo artifacts surround the periphery of the cell membranes and are also evident on some of the larger organelles within the cells. Although many of the internal cellular details are present at high resolution in this image, information about intracellular contracts is suppressed by the bright (halo) regions appearing between adjacent cell membranes. The absence of halo artifacts is evident in the same viewfield (Figure 4(d)) when imaged with differential interference contrast. Internal details of the cells are less evident in DIC, but the periphery of the cellular membranes is imaged more clearly than with phase contrast. In fact, the close proximity of neighboring cells is very obvious in the DIC image, which renders the technique more accurate for studying intercellular events, such as contact inhibition.

Members of the filamentous green algae genus, Zygnema, exhibit an elongated, linear structure composed of repeating individual cellular units. In phase contrast (Figure 4(e)), filamentous Zygnema specimens display a pronounced halo surrounding the exterior of the rod-like algae filaments, but also contain substantial internal detail that is obscured by the halo artifact. The bisymmetrical U-shaped repeating internal cellular structures (Figure 4(e)) give rise to large halos that significantly reduce contrast and mask smaller features. Differential interference contrast eliminates the halo artifacts present in phase contrast images of Zygnema (Figure 4(f)), and reveals substantially more of the internal detail present in the center of the filament. However, in order to obtain the most definitive conclusions regarding Zygnema filament structure, the two imaging techniques should be applied in conjunction (as is the case for many of the specimens described above).

As discussed above, specimen features having a higher refractive index than the surrounding medium display bright halos around the periphery and exhibit darker central portions when imaged with positive phase contrast optical systems. The opposite effect occurs when features having lower refractive index than the medium are observed (dark halos and light internal regions). Halo effects vary with the size of the specimen feature and are heavily dependent upon the refractive index differential between the specimen and the surrounding medium. Larger differences in refractive index tend to generate more pronounced halo effects, which are also dependent on instrumental parameters, such as phase plate dimensions and neutral density, and the objective magnification factor. In addition, specimen feature size plays a paramount role in determining the severity of halo artifacts in phase contrast microscopy.

The situation is quite different in DIC microscopy, where large refractive index differentials and specimen feature size do not impair image quality. In fact, pronounced differences of refractive index between the specimen and its surrounding medium are often highly desirable, and usually lead to excellent images in differential interference contrast. Feature size does not generally present a problem in DIC microscopy. Specimens having large fluctuations in size and refractive index can frequently be imaged simultaneously (side-by-side) with differential interference contrast optical systems to yield structures having excellent contrast, which enables even minute details to be observed and recognized.

Halo artifacts and image contrast are also affected by the optical thickness of the specimen to a much larger degree in phase contrast than in DIC microscopy. At large optical path differences (up to half a wavelength), ambiguous phase contrast images can be produced because the thickest features in a specimen often display contrast levels that are very close, or identical, to those of the thinnest features. This arises from the complex relationships between optical path length (specimen thickness and refractive index), contrast, and phase angles. In general, ideal phase contrast specimens should not have features exceeding about one-tenth wavelength in optical path difference. Thin specimens are also desirable for DIC microscopy, but the technique is capable of producing suitable images with specimens that have much larger thickness values (and optical path lengths) than are recommended for phase contrast. However, very thick specimens can negatively influence the overlap of interference planes between the objective and condenser prisms, reducing the quality and contrast in DIC images.

In flat, extended specimens of uniform optical path length, an artifact known as shade-off is commonly observed in phase contrast microscopy. Shade-off is manifested by the central portion of the specimen displaying a gradual decrease in contrast until the intensity approaches that of the surrounding medium. In extreme cases, extended specimens will only be visible in phase contrast due to the presence of their halos. In DIC, a variation in image intensity only occurs if there is a marked change in optical path difference, and the boundaries of extended specimens are often the only feature that can be recognized with this technique.

Depth of Field and Optical Sectioning

Because phase contrast images can become ambiguous (or even experience a reversal of contrast) when the specimen optical path length fluctuates over a large range, the technique should be restricted to specimens having a path difference of one-tenth wavelength or less, as previously discussed. In other words, thick specimens, or those having wedge-shaped structures, are generally not suitable for quantitative investigations with phase contrast microscopy. Thin specimens also tend to produce superior images in differential interference contrast, but thicker specimens (having an optical path difference between one-tenth and a full wavelength) can also be imaged at wide apertures using optical sectioning techniques.

The illumination aperture in phase contrast is fixed by the size of the condenser annulus and cannot be varied to achieve increased or decreased depth of field. This restriction hampers observations of specimens containing significant halo artifacts, which can sometimes obscure focused specimen features due to overlapping details originating from excessive light originating outside the objective focal plane. Such problems often arise when attempting to image specimens that are too thick for phase contrast. However, as previously discussed, the optical sectioning capability of differential interference contrast microscopy (at wide condenser aperture sizes) enables excellent images to be obtained with relatively thick specimens.

The benefits of large aperture optical sections from thick specimens in differential interference contrast are illustrated in Figure 5, and compared to the same viewfields imaged with phase contrast. Figure 5(b) illustrates a medusa bud from a reproductive polyp of the Obelia hydrozoan imaged at high magnification in DIC with the condenser iris diaphragm opening set to approximately 95 percent of the objective aperture size. The resulting thin optical section reveals sharply focused image details of the medusa features, which are only minimally obscured by light originating away from the focal plane. When the same viewfield is examined in phase contrast (Figure 5(a)), the medusa image is blurred by halos and exhibits reduced resolution due to the restricted aperture of the optical system.

Similar situations are presented in Figures 5(c) through 5(f). Figure 5(c) depicts overlapping wing scales from the monarch butterfly (Danaus plexippus), one of the most common butterflies in North America. Individual scales are not distinguishable in Figure 5(c) because of halo artifacts and indistinct features that are not in the plane of focus. In general, the image is confused by numerous artifacts, making specimen features difficult to decipher. Imaging the same specimen in differential interference contrast (Figure 5(d)) eliminates many of the distracting artifacts positioned away from the focal plane and clearly delineates the surface features in a single wing scale. Likewise, an egg packet within the abdomen of a canine cucumber tapeworm (Dipylidium caninum) reveals sharp edges surrounding individual eggs when imaged in DIC (Figure 5(f)). However, the focused eggs are devoid of distinctive structure in phase contrast (Figure 5(e)), primarily due to halos from others eggs situated away from the focal plane.

The optical sectioning capability of differential interference contrast at large condenser aperture sizes and high numerical aperture is critical for imaging single focal planes in thick phase specimens. Details and stray light from features lying outside of the immediate focal plane do not compromise DIC images in the same manner as phase contrast images, which are often complicated by halo artifacts. As a result, differential interference contrast can be employed to obtain excellent images under somewhat unfavorable conditions (very thick specimens), even when large depth of field renders the identification of phase structures impossible in phase contrast microscopy due to overlapping details.

Birefringent Specimens

An advantage of phase contrast over DIC is the ability to successfully image dichroic and birefringent specimens. Because differential interference contrast optical systems rely on polarized light to establish the orientation of wavefront fields, differential absorption of ordinary and extraordinary waves by a birefringent or dichroic specimen leads to confusing images. Phase contrast does not require the use of polarized light, and is free of optical disturbances generated by birefringent specimens.

One of the primary concerns pertaining to birefringence artifacts in differential interference contrast microscopy arises from the widespread use of plastic tissue culture vessels. Stress inherent in the molded polymer vessel structure produces excessive birefringence that depolarizes light and confuses the interpretation of DIC images. Thus, living cells grown in these containers cannot be imaged satisfactorily with differential interference contrast and, instead, are usually observed and photographed with phase contrast or Hoffman modulation contrast optical systems.

Typical artifacts arising from imaging birefringent specimens in DIC are presented in Figure 6, along with images of identical viewfields in phase contrast. Starch storage granules (appearing as globules) from a quadruple-stained thin section of potato (Solanum tuberosum) tissue, also illustrating a portion of the mature tuber with periderm, cortex, and reduced vascular tissues, are presented in Figures 6(a) and 6(b). Because carbohydrates in potato starch grains display an ordered lamellar molecular structure, portions of the grains (and in some cases, the entire grain itself) are birefringent and absorb the polarized wavefronts that leave the condenser Nomarski prism and pass through the specimen. As a result, the potato starch grains observed in DIC microscopy exhibit interference colors and the characteristic Maltese cross patterns (originating from the crossed polarizers), which are typical of birefringent anisotropic specimens having spherical symmetry (Figure 6(b)). The birefringence artifact in DIC masks much of the internal structure of the starch grains, which reveal far more detail when imaged in phase contrast (Figure 6(a)), even though the specimen has been stained.

Illustrated in Figure 6(c) is a phase contrast image of a single rayon fiber showing the pitted morphology that is present both on the surface and inside the polymer. Rayon is fabricated from cellulose, cotton linters, or wood chips by treating the bulk materials with sodium hydroxide and carbon disulfide, and then passing the resulting viscous solution through spinnerets. The fibers produced in this process have long-range order and are usually very highly birefringent. DIC examination (Figure 6(d)) of the rayon fiber confirms the birefringent character, which is manifested in Newtonian interference colors present in the image. The colors, while beautiful, obscure specimen detail and decrease the usefulness of DIC for imaging materials of this type. The birefringent images illustrated in Figures 6(b) and 6(d) were recorded using relatively thin (5-20 micrometer) specimens at low magnification (20x) that remain in sharp focus throughout the viewfield. When thicker birefringent specimens are imaged with DIC, the interference colors originating from planes away from the point of focus tend to obscure specimen detail to a much greater degree. Therefore, as specimen thickness increases (for birefringent specimens), the resulting images produced in DIC tend to lose contrast and the quality deteriorates rapidly.

The problems associated with observing living cells grown in plastic tissue culture vessels using differential interference contrast microscopy are clearly evident by comparing Figures 6(e) and 6(f). Several Indian Muntjac (Muntiacus muntjak) deer skin fibroblast cells in monolayer culture are illustrated in Figure 6(e), imaged using a long working distance objective and condenser combination in phase contrast on an inverted tissue culture microscope. The cells were grown in molded polystyrene vessels having an upper and lower wall thickness of approximately 1.2 millimeters each (combined thickness of polystyrene is 2.4 millimeters). Even with phase contrast objectives having correction collars that are designed for observation through thick glass or plastic, the image quality suffers when compared to cells imaged on thin (170 micrometer) cover glass plates (compare Figure 6(e) to Figure 4(c)). However, much of the internal cellular detail is still visible, including nuclei, the nucleolus, vacuoles, and numerous smaller particles.

In differential interference contrast, the cultured Indian Muntjac fibroblast cells illustrated in Figure 6(e) lose a significant amount of contrast (Figure 6(f)) due to absorption and interference effects from the birefringent polystyrene container. Because the container walls are relatively thick, strain birefringence depolarizes the ordinary and extraordinary wavefronts in the upper wall of the tissue culture vessel before they pass through the cells, and once again after exiting the cells and passing through the lower wall. The result is a drop in contrast so severe, the cells can be imaged only when the condenser diaphragm is almost completely closed (similar to brightfield techniques with transparent specimens). Under these conditions, images display numerous diffraction artifacts in addition to the low contrast, and render DIC practically useless for this type of observation.

The major source of errors in examining birefringent specimens with differential interference contrast microscopy arises from the necessity of using polarized light, as discussed above. In birefringent specimens, the polarized orthogonal (ordinary and extraordinary) wavefronts are absorbed to different degrees, depending upon their orientation with respect to anisotropic molecules within the specimen. As a result, when the orthogonal wavefronts are recombined in the objective Nomarski prism and pass through to the analyzer, they interfere with different intensity. The final image, therefore, is not only a function of the optical path difference experienced by the two wavefronts (normally a critical factor in DIC), but also the differential amplitudes of the two beams induced by the specimen. The effect on images is similar to a microscope configuration in which the transmission axes of the polarizer and analyzer are not perfectly perpendicular (in effect, slightly uncrossed). Because phase contrast does not rely on polarized light, the technique is largely free of artifacts induced by birefringent specimens.

Stained Specimens

Lightly stained amplitude specimens, which are not necessarily birefringent but may retain a significant amount of phase gradient character, experience reduced intensity due to absorption of visible wavelengths by the objective phase plate in phase contrast microscopy. However, these specimens can often produce satisfactory results in differential interference contrast when substantial optical path gradients are present and both polarized wavefronts are equally absorbed. In fact, selective staining of intracellular organelles (for instance, intact nuclei and metaphase chromosomes) can be coupled to imaging with DIC microscopy in order to enhance specimen detail and segregate events occurring at the time the cells were fixed.

Illustrated in Figure 7 are several examples of specimens stained with common biological (visible light absorbing) chromophores, and imaged with both phase contrast and DIC microscopy. A thin section of human fat-stained adipose tissue observed in differential interference contrast is presented in Figure 7(a), and the same viewfield with phase contrast in Figure 7(b). The phase contrast image is confusing and lacks resolution of fine detail, primarily because of halo artifacts that mask the advantages of the biological stain. Phase contrast is clearly not appropriate for retrieving any significant information from this specimen. In distinction, a corresponding DIC image of the same specimen (Figure 7(a)) renders the fat globules in shadow-cast pseudo three-dimensional relief, which distinguishes them quite well from other features in the image.

The same effect is observed with DIC microscopy in Figure 7(c), where differentially stained meiotic nuclei and chromosomes within a frog testes thin section can be observed in high contrast. This image benefits from the clever application of multiple stains that aid in chromatically segregating the genetic material from other intracellular components. In fact, the specimen yields more significant information when imaged in DIC than it does in the target observation mode of brightfield illumination. The same viewfield in phase contrast (Figure 7(d)) is confused by halos surrounding the densely stained intracellular components, which are far more difficult to distinguish in this imaging mode. A similar situation exists for a mammalian tooth stained thin section presented in Figures 7(e) and 7(f). In DIC, striated lamella in the enamel are easy to observe due to their relief (Figure 7(e)), while less than optimal optical path differences coupled to overstaining mask these structures in phase contrast (Figure 7(f)). The examples in Figure 7 provide strong evidence that differential interference contrast is a suitable technique for observing lightly stained specimens, even though the same specimens are difficult to image in phase contrast.

Conclusions

The most obvious difference between DIC and phase contrast microscopy is the pseudo three-dimensional shadow-cast images formed by differential interference contrast optical systems. Specimen images are rendered in a manner reminiscent of the shadowing techniques utilized for many years in electron microscopy, which yield information on topographical properties of the specimen. However, in DIC, the apparent hills and valleys in images must not be mistaken as an indication of actual topographical profile in the specimen, and should always be interpreted with caution. Phase contrast does not produce images having significant three-dimensional character.

Many of the similarities and differences between phase contrast and differential interference contrast microscopy are summarized in Table 1 for a number of variables, including image brightness, resolution, phase shift limitations, artifacts, specimen characteristics, and cost. In summary, image brightness in both phase contrast and DIC is much less (only a few percent) than ordinary brightfield observation, but these contrast-enhancing techniques are capable of providing far more highly defined images. Illumination is more critical for DIC microscopy than phase contrast, especially when using low transmission polarizers at high magnifications. Often, a 100-watt tungsten-halogen or mercury arc-discharge lamp is required for satisfactory imaging, whereas phase contrast microscopes perform adequately with 50-watt incandescent lamps. The lateral (x-y) and axial (z) resolution in differential interference contrast microscopy dramatically exceeds that with phase contrast, which exhibits modest or poor resolution in both dimensions even though the phase shift detection limit is similar in both cases.

Characteristics of Phase Contrast and DIC Microscopy

Characteristic

Phase Contrast

DIC

Image Brightness

(Brightfield = 100 Percent)

1.3 Percent

0.36 - 2.3 Percent

Epi-Fluorescence Light Loss

(Brightfield = 0 Percent)

28 Percent

73 Percent

Lateral Resolution

Condenser

Annulus

Restricted

Superior

Axial Resolution

(Depth Discrimination)

Poor

Superior

Illuminating Aperture

10 Percent of

Objective NA

Variable

Phase Shift

Detection Limit

< l/100

< l/100

Utility at High Phase Shifts

Not Useful

Useful

Azimuthal Effects

No

Yes

Halos and Shade-Off

Yes

No

Stained Specimens

Not Useful

Useful

Birefringent Specimens

Useful

Not Useful

Birefringent Specimen

Containers

Yes

No

Brightfield Image

Deterioration

Slight

None

Cost

Moderate

High

Table 1

Thick phase specimens having large optical path differences can be readily imaged in differential interference contrast using optical sectioning techniques, but are often difficult to image with phase contrast due to halo artifacts. Other factors, such as azimuthal effects and the ability to image stained specimens in DIC, help to offset the two techniques. Phase contrast is far more useful for routine observations in tissue culture laboratories, where imaging living cells in plastic culture vessels is a daily activity. Alternatively, high resolution information is best retrieved using differential interference contrast methods (often coupled to time-lapse video) for live cell imaging. In general, DIC requires more technical skills to configure and operate, including the complex alignment process, continuous manipulation of specimen focus, aperture iris diaphragm adjustment, specimen orientation, and lateral displacement of the objective Nomarski prism to achieve optimum contrast. Phase contrast microscopes are far easier to align and operate, and can be utilized quite effectively by relatively inexperienced microscopists.

Phase contrast is generally limited in utility to imaging transparent phase specimens having poor contrast. Conversely, DIC microscopy can be employed both for stained and unstained specimens, but suffers from artifacts when birefringent features are encountered. More control over specimen contrast is afforded by translating the objective prism (or de Sénarmont compensator) in DIC, which can be adjusted over a wide range of positions to achieve varying levels of grayscale interference colors, or multi-colored optical staining effects at large path differences. In addition, the condenser aperture diaphragm can also be adjusted in DIC to alter specimen contrast and resolution. Phase contrast is limited to the contrast afforded by the condenser annular diaphragm and the objective phase plate housed in the objective. Virtually no adjustment of contrast (other than exchanging phase plates) is possible with phase contrast microscopy.

Among the most important considerations in many laboratories, when comparing DIC and phase contrast, is the cost of accessory components. Because DIC requires several expensive birefringent Nomarski prisms and strain-free optical elements (objectives and condenser), the instrumentation is significantly more expensive than phase contrast components. In many cases, particularly when photomicrography and/or digital imaging is not a primary consideration, phase contrast will often serve the purposes of a laboratory. However, when high-resolution images must be obtained from living cells and tissues, or similar fragile transparent phase specimens, differential interference contrast is the technique of choice.

In general, phase contrast and differential interference contrast microscopy should be considered as complementary (rather than competing) techniques, and employed together to fully investigate specimen optical properties, dynamics, and morphology. The introduction of modern phase contrast objectives having phase plates of lower neutral density enables the microscopist to, in many cases, utilize a single set of objectives for brightfield, fluorescence, phase contrast, and DIC imaging.

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.

Fluorescence Microscopy

Combination Methods

with

Differential Interference Contrast (DIC)

To minimize the effects of photobleaching, fluorescence microscopy can be combined with other techniques that are non-destructive to the fluorochrome, such as differential interference contrast (DIC), Hoffman modulation contrast (HMC), transmitted darkfield illumination, and phase contrast.

The idea is to locate a specific area of interest in a specimen using the non-destructive contrast enhancing technique then, without relocating the specimen, switch the microscope to fluorescence mode. The results of a typical experiment of this type are illustrated in Figure 1. Figure 1(a) illustrates a rat retina optic ganglion tissue thin section imaged using differential interference contrast. The photomicrograph in Figure 1(b) shows the same viewfield, but this time imaged using fluorescence illumination (a mercury vapor lamp and an Olympus WU filter cube) with cells stained by the fluorochrome fast blue, a diazonium salt that specifically stains phospholipids in the myelin sheath. Figure 1(c) illustrates the two techniques used in combination to produce a beautiful photomicrograph of fluorescent-stained optic ganglion lipid tissue superimposed on a differential interference contrast image of the retina. This image was recorded with a specialized fluorite objective designed to permit simultaneous observation of both fluorescence and differential interference contrast with the same objective.

The microscope configuration typically utilized to simultaneously image specimens using both differential interference contrast (DIC) and fluorescence illumination is illustrated in Figure 2. DIC is conducted using transmitted light provided by a tungsten-halogen lamp positioned in a lamphouse attached to the microscope base. Light passing through the field diaphragm is reflected by a mirror into the substage condenser and through a Wollaston prism located at the front focal plane of the condenser.

Interactive Tutorial

Fluorescence Combination Microscopy

Explore combinations of fluorescence microscopy with additional contrast-enhancing techniques using both phase contrast and differential interference contrast (DIC) methods.

Light diffracted by the specimen is first passed through a second Wollaston prism positioned near the objective rear focal plane before interfering at the intermediate image plane with light passing through the specimen undiffracted. Simultaneously, ultraviolet light emitted by a mercury burner is passed through an exciter filter, then reflected by a dichroic mirror onto the specimen from above. Secondary fluorescence emitted by the chromophore attached to the stained specimen is captured by the objective and passed through the barrier filter and into the eyepieces and/or phototube. This configuration can be used to image specimens using the techniques (fluorescence and differential interference contrast) individually or in combination.

Contributing Authors

Mortimer Abramowitz - Olympus America, Inc., Two Corporate Center Drive., Melville, New York, 11747.

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

Differential Interference Contrast

Interactive Tutorials

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.

Contributing Authors

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.

Differential Interference Contrast

Interactive Tutorials

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.

The tutorial initializes with a randomly chosen specimen image appearing in the left-hand window, and a pair of diagonal blue lines slowly traversing the specimen portion of the image at a 45-degree angle. The shear axis (x) of the Nomarski prisms in the differential interference contrast microscope is indicated by the double-headed white arrow located in the lower corner of the specimen image window (Note: the shear axis of the microscope is parallel to the diagonal lines). As the diagonal blue lines travel across the image, the amplitude profile of the specimen corresponding to a thin diagonal slice between the two blue lines is plotted in the DIC Amplitude Profile window. In addition, the optical path difference (OPD) along the diagonal slice is presented in the Optical Path Difference window.

In order to operate the tutorial manually, first click on the Pause button to halt the motion of the double blue lines. Next, place the mouse cursor on the lines, and then click and drag the lines across the image to view amplitude profiles and optical path difference variations at any slice along the specimen surface. In this manner, variations in amplitude profile and the relationship to the corresponding optical path difference can be examined and compared at each point on the specimen and surrounding background. After a specimen has been thoroughly studied, use the Choose A Specimen pull-down menu to select a new specimen having a different profile. The Applet Speed slider can be utilized to alter the speed at which the blue diagonal lines automatically traverse the specimen image.

After orthogonal wavefronts generated by the beamsplitting prism pass through a specimen phase gradient, they are recombined through differential interference by a second prism and an analyzer (another polarizer) to yield a high-contrast rendition of the gradient. Both differential interference contrast and phase contrast rely on specimen phase differences between sampling and reference beams to produce an image based on varying amplitude. Phase contrast translates image amplitude information from the phase variations exhibited between light waves diffracted by the specimen and a reference beam that passes through the condenser annulus, specimen, and phase plate. In distinction, however, the DIC image corresponds to the mathematical first derivative, rather than the magnitude, of the gradient profile obtained from the specimen optical path difference.

The relationship between optical path gradients and intensity profiles in DIC microscopy is illustrated in Figure 1. The specimen presented in Figure 1(a) is a doughnut-shaped human erythrocyte imaged at high magnification in differential interference contrast with the shear axis indicated by a double-headed arrow (northwest to southeast). A plot of the optical path difference (the ordinate) versus a cross section of the erythrocyte along the shear axis (the abscissa) is shown in Figure 1(b). Note that the optical path profile reflects the thin center and thick edges exhibited by human blood cells. An intensity scan across the differential interference contrast image, which corresponds very closely to the first derivative of the optical path difference curve (Figure 1(b)) when added to a constant, is presented in Figure 1(c). Positive and negative slopes in the erythrocyte optical path profile generate regions of higher and lower amplitude, respectively, in the first derivative scan and corresponding differential interference contrast image. Regions of the optical path difference profile that exhibit no change in slope have the same intensity as the background and correspond to the baseline in the first derivative plot of the optical path difference.

Contributing Authors

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.

Jan Hinsch - Leica Microsystems, Inc., 110 Commerce Drive, Allendale, New Jersey, 07401.

Matthew J. Parry-Hill, Robert T. Sutter, and Michael W. Davidson - National High Magnetic Field Laboratory, 1800 East Paul Dirac Dr., The Florida State University, Tallahassee, Florida, 32310.

Differential Interference Contrast

Interactive Tutorials

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 translating a Nomarski or Wollaston prism affect the optical path difference and resulting specimen intensity in a DIC microscope.

The tutorial initializes with a model of an erythrocyte (human red blood cell) oriented parallel to the shear direction of the incident wavefront and viewed in cross section on the lower left-hand side of the window. The specimen is assumed to be homogeneous, isotropic, and non-birefringent, having a refractive index (n(s)) greater than that of the surrounding medium (n(m)). A sheared wavefront (located beneath the specimen in the window) is created by passage of plane-polarized light through a Wollaston (or Nomarski) prism housed at the condenser front focal plane (not illustrated). The extraordinary wavefront is drawn as a black line and the ordinary wavefront is represented by a red line displaced slightly to the right. For clarity and ease of illustration, both wavefronts are illustrated parallel to the browser window, but in reality, they are oriented mutually perpendicular to each other.

The wavefronts first pass through the specimen to produce an optical path difference created by the refractive index variations between the specimen and its surrounding medium. In the case of a transparent specimen, the optical path difference is determined by the product of the refractive index and the geometrical thickness of the specimen. The portions of the wavefronts passing through the specimen are retarded relative to those that pass through adjacent portions of the surrounding medium.

After leaving the specimen, the wavefronts enter a second Wollaston (or Nomarski) prism that is located in the objective rear focal plane. The function of this prism is to cancel the angular wave splitting (shear) induced by the first prism located before the specimen in the condenser front focal plane. Upon initialization, there is no additional optical path difference introduced by the second Wollaston prism. However, a lateral displacement of the wavefront deformation occurs as shear is eliminated by the objective Wollaston prism. This produces a path difference introduced between the wavefronts in regions where the specimen exhibits large phase gradients. Under these conditions, the optical system of the microscope is in a condition termed maximum extinction, and the specimen image (presented in the DIC Specimen Image window) appears as brightly highlighted regions on a very dark or black background.

For paraxial light rays, the ordinary and extraordinary wavefronts leave the Wollaston prism in phase and arrive at the specimen still in phase. The wavefronts pass through the surrounding medium and regions of the specimen having equal thickness (no refractive index gradients) without introduction of retardation between the waves. In regions (sloped specimen areas) having refractive index gradients, one or both of the wavefronts are retarded when passing through the specimen. At the left specimen edge, the ordinary wavefront is retarded to a greater degree than is the extraordinary wavefront, while the opposite is true for the right specimen edge.

Upon entering the analyzer, the phase differences in the wavefronts result in interference that produces the intensity and contrast variations observed in the microscope eyepieces. The intensity distribution (I) of the differential interference contrast image is given by the equation:

I = Ip • sin^2((dc + ds/2) + Ic

where I(p) is the light intensity that would be observed if the analyzer and polarizer transmission azimuths (vibration directions) were parallel, d(c) and d(s) are the phase retardations between the ordinary and extraordinary wavefronts induced by the second Wollaston prism and specimen respectively, and I(c) is the light intensity when the analyzer and polarizer are crossed and d equals zero. The term I(c) represents stray light in the optical system introduced by a variety of artifacts including birefringence in lens elements, rotation of the polarization plane by highly curved lens surfaces, and imperfect polarization of light by the polarizer and analyzer. The intensity profile of the specimen across the shear axis of the microscope is plotted as a function of lateral wavefront displacement in the upper left-hand corner of the applet window (see the graph labeled Intensity). Adjacent to this graph is a digital image of a typical specimen observed under these conditions (the DIC Specimen Image window).

Specimens produce contrast based primarily on gradients in optical path that occur in the direction of shear in differential interference contrast microscopy. This gradient-dependent contrast generation mechanism occurs because the ordinary and extraordinary wavefronts are separated by less than the optical resolution of the microscope. Regions having constant optical path will produce minimal or no contrast while regions exhibiting large gradients and optical path differences tend to produce images of high contrast. Image contrast is at a maximum in the shear direction between the wavefronts and is minimal in regions perpendicular to the shear direction. As can be seen in the tutorial specimen image, intensity and contrast are very poor at maximum extinction (no bias retardation applied to the optical system). Under these conditions, edges and gradients appear brighter than the background and the overall image assumes the profile of a darkfield image produced by a thin specimen (a bright image on a dark background).

Changes in bias retardation can be introduced by translating the Bias Retardation slider to the left or right. Moving the slider in either direction increases the optical path difference between the ordinary and extraordinary wavefronts and produces a corresponding change in image intensity. When the slider is translated to the left (Positive Bias), the ordinary wavefront is retarded, producing an increase in the optical path difference. In contrast, moving the slider to the right (Negative Bias) retards the extraordinary wavefront in relation to the ordinary wavefront. In both cases, an inversion of brightness distribution is brought about by the optical path differences. A new specimen can be selected using the Choose A Specimen pull-down menu. The oil drop specimens simulate situations where the specimen refractive index is either higher (High RI) or lower (Low RI) than that of the surrounding medium.

Contributing Authors

Mortimer Abramowitz - Olympus America, Inc., Two Corporate Center Drive., Melville, New York, 11747.

Jan Hinsch - Leica Microsystems, Inc., 110 Commerce Drive, Allendale, New Jersey, 07401.

Edward D. Salmon - CB# 3280, Coker Hall, The University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599.

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

Differential Interference Contrast

Interactive Tutorials

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.

The tutorial initializes with two coherent wave packets of white light, which are represented by gray sine waves (initially the sine waves are overlapped, so only a single wave appears in the window). One wave is light gray in color while the other wave is dark gray. In order to reduce the complexity of the tutorial (and to clarify explanation of the concept) only three component wavelengths of white light are employed to represent the entire spectrum: Blue (400 nanometers), Green (550 nanometers), and Red (700 nanometers). Each colored wave in the tutorial (blue, green, and red) represents the resultant wave that occurs from either constructive or destructive interference of the coherent gray waves. A reference point on the abscissa (the Phase axis) denoted by S(1) represents the relative wavefront phase position when no optical path difference is applied to the system.

In order to operate the tutorial, use the mouse cursor to shift the Optical Path Difference (OPD or D) slider to the right from its default far left position. Changing the value of this slider mimics the effects of translating the objective Nomarski prism across the optical path in order to generate bias retardation in a DIC microscope. As the slider is shifted to the right, the induced optical path difference is presented above the slider (in nanometers) and also in the tutorial window as an expanded path length between the reference wavefront (S(1)) and the retarded (or advanced) wavefront (S(2)), which occurs when bias retardation is added to the optical system. In addition, Michel-Lévy colors produced by interference of the recombined wavefronts at the microscope analyzer are presented in the lower right-hand side of the tutorial window (the Interference Color box). The range of the Optical Path Difference slider is zero (no bias retardation) to 600 nanometers.

At small optical path differences (less than 140 nanometers) the amplitude of the red, green, and blue wavefronts is larger than the individual gray wave packets, a phenomenon that results from constructive interference. Between 140 and 275 nanometers, destructive interference effects take over and the amplitude of recombined waves is decreased. When the optical path difference is equal to a whole or multiple of a single white light component wavelength (in the tutorial, either 400, 550, or 700 nanometers), then both the light and dark gray sine waves have the same phase and constructive interference doubles the amplitude for that wavelength. However, at values equaling half of a component wavelength (200, 275, and 350 nanometers), the two wave packets (gray and light gray sine waves) are 180 degrees out of phase, resulting in complete destructive interference. Thus, at 200 nanometers, for example, white light is deprived of the blue component (having a wavelength of 400 nanometers). The same results occur for the green and red components at 275 and 350 nanometers, respectively. When one of the primary components is missing from white light, the remaining wavelengths are mixed uniformly according to their amplitude ratios to yield a unique interference color.

Because the amplitude ratio of the white light components varies with optical path difference, each path difference is associated with a characteristic interference color, which can be determined by examining a Michel-Lévy color chart. As discussed above, translating the objective Nomarski prism across the microscope optical axis produces a constant optical path difference between wavefronts traveling through the optical system from the condenser prism to the objective prism. Introduction of this optical path difference results in the generation of interference colors when vector components from recombined wavefronts are added together at the microscope analyzer. These are the colors observed for the background in the microscope viewfield either with or without a specimen positioned on the stage (Note: an optical path difference introduced by the specimen will simply add to the one created by the microscope). A path difference of 50 nanometers produces a hue between iron and lavender gray. Likewise, optical path differences of 200, 400, and 600 nanometers yield interference colors that have been named clear gray, brown-yellow, and indigo, respectively.

Contributing Authors

Jan Hinsch - Leica Microsystems, Inc., 110 Commerce Drive, Allendale, New Jersey, 07401.

Matthew J. Parry-Hill, Robert T. Sutter, and Michael W. Davidson - National High Magnetic Field Laboratory, 1800 East Paul Dirac Dr., The Florida State University, Tallahassee, Florida, 32310.

Differential Interference Contrast

Interactive Tutorials

Specimen Orientation Effects on DIC Images

In differential interference contrast (DIC) 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.

The tutorial initializes with a randomly chosen specimen appearing in the virtual DIC microscope viewport, where the shear axis is oriented at a 45-degree angle (from the upper left to the lower right of the field) with respect to the browser window. In order to operate the tutorial, use the mouse cursor to translate the Specimen Orientation slider over a 360-degree range. As the specimen is rotated, azimuthal effects will be observed in specimen amplitude, with light areas growing darker and vice versa. In many cases (especially with highly asymmetric and elongated specimens), overall image contrast will fluctuate as a function of specimen orientation, and some features may become indistinguishable from the background. In order to select a new specimen, use the Choose A Specimen pull-down menu.

Each specimen examined in differential interference contrast microscopy will have an optimum bias retardation setting that generates a maximum level of contrast in the final image. Very thin specimens that display a shallow refractive index gradient, such as living cells in culture, generally benefit from equally low bias settings only slightly larger than the largest phase shift present in the specimen (on the order of approximately one-twentieth of a wavelength, or 30 nanometers). However, thicker specimens often require higher bias settings (up to 90 degrees) at large condenser apertures to produce satisfactory results, usually through optical sectioning. Because many specimens are composed of features displaying a variety of different sizes and refractive indices, the optimum bias retardation setting is usually a compromise.

Azimuthal effects of orientation phenomena in differential interference contrast for several specimens are presented in Figure 1. In all cases, the shear axis is directed northwest to southeast (similar to the orientation in the tutorial viewport), but is not indicated on the individual digital images. Figure 1(a) and 1(b) illustrate the periodic spacing of pores and striae exhibited by a mounted frustule from the diatom Gyrosigma attenuatum. When the long axis of the frustule is oriented perpendicular to the shear axis (Figure 1(a)), the pores appear to merge into a series of closely spaced ridges and are not resolved individually. In contrast, reorientation of the frustule in a direction parallel to the shear axis reveals the knob-like geometry of pore structure in this species.

The thorax region containing gill ribs of a freshwater Daphnia (water flea) is presented in Figure 1(c). In this orientation, individual rib structures are clearly visible (perpendicular to the shear axis) and joined to a common spine, which appears as a long series of knobs parallel to the shear axis. When the specimen is rotated 90-degrees (Figure 1(d)), much of the rib structure loses contrast, and the spinal knobs merge into a single ridge. Finally, inclusions in a ctenoid fish scale lack contrast and are difficult to distinguish when superimposed over the striated body spines (Figure 1(e)). Rotating the specimen to bring the spines parallel to the shear axis (Figure 1(f)) reduces their contrast and renders the inclusions clearly visible.

A number of factors should be considered when adjusting bias retardation to produce optimum specimen contrast. The level of bias that must be introduced to maximally darken one slope or edge of the specimen also yields the maximum possible contrast between the specimen and the background. Therefore, for each specimen, one particular objective prism (or de Sénarmont compensator) setting will usually introduce the greatest degree of contrast. When this value is exceeded, contrast will decrease. Thicker specimens, which often suffer from light scattering artifacts, usually require a larger bias retardation setting (up to a full wavelength) than thinner specimens to obtain extinction in regions having significant phase gradients. Finally, when the iris diaphragm opening size in the condenser exceeds 75 percent of the objective rear aperture, contrast is reduced as a result of excessive light scattering in the optical system. With this difficulty in mind, use caution when opening the condenser aperture to perform optical sectioning experiments.

Contributing Authors

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.

Jan Hinsch - Leica Microsystems, Inc., 110 Commerce Drive, Allendale, New Jersey, 07401.

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

Differential Interference Contrast

Interactive Tutorials

Wavefront Shear in Wollaston and Nomarski Prisms

Explore how Wollaston and Nomarski prisms act as a beamsplitter to separate or shear a polarized beam of light into two coherent and orthogonal components that pass through and interact with slightly different areas of a specimen in differential interference contrast (DIC) microscopy. This interactive tutorial examines differences between the location of the interference plane in both prism types, and how the position of the plane can be varied with changes to the optical axis orientation in a single prism wedge.

The tutorial initializes with a standard Wollaston prism appearing in the window and a beam of linear (plane) polarized light entering through the bottom portion of the prism at a 45-degree angle. As the linearly polarized wave enters the lower portion of the Wollaston prism, it is split (or sheared) into two plane-polarized components that are oriented mutually perpendicular (orthogonal) to each other. One of the waves is designated the ordinary (O) wave and vibrates in a direction perpendicular to the optical axis of the prism, while the other is termed the extraordinary (E) wave with a vibration direction parallel to the prism optical axis. Ordinary wavefronts are represented by blue bars as they progress through the Wollaston prism, and extraordinary wavefronts are characterized by red bars. In addition, the optical axes of the individual prism wedges are indicated by an arrow (parallel to the browser window) in the lower wedge, or a bull's-eye target (perpendicular to the browser window) in the upper wedge. The interference plane is represented by a dashed line.

In order to operate the tutorial, use the Prism Position slider to translate the compound prism back and forth (to the left and right) across the incident polarized light beam. As the prism is moved to the right, the beam travels a greater distance through the lower prism half, affecting the relationship between the emerging ordinary and extraordinary wavefronts. When the prism is moved to the left, the beam travels through only a short distance in the lower prism, while traversing a larger portion of the upper wedge.

Translating the Prism Type slider to the left produces a change in the orientation of the optical axis in the lower prism wedge, and also transforms the compound prism from a Wollaston to a Nomarski design. Simultaneously, the position of the interference plane is shifted from the central region of the compound prism, first into the upper wedge, and eventually to the exterior of the prism. As the prism type is altered, the trajectories of the ordinary and extraordinary wavefronts are modified to reflect how each of these orthogonal components traverse the prism.

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. The polarizer in a DIC microscope (positioned beneath the Wollaston prism) is oriented so that linearly polarized light enters the prism at a 45-degree angle with respect to the optical axes of the two birefringent prism halves.

Each of the sheared wavefronts experiences a slightly different refractive index that varies with the composition of the Wollaston prism. Prisms made of quartz, which is a positive uniaxial crystal, display a refractive index difference on the order of 0.6 percent. Because propagation speed of the waves through the crystal is inversely proportional to the refractive index, each wave travels at a slightly different velocity. In quartz, the ordinary ray travels faster than the extraordinary wave due to a slightly lower refractive index. Alternatively, in negative uniaxial crystals (such as calcite), the extraordinary wave experiences a lower refractive index and propagates faster than the ordinary wave.

In the tutorial, each wavefront is represented by either a blue (ordinary) or red (extraordinary) bar, as previously discussed. Because the virtual Wollaston prism in this tutorial is composed of quartz, the ordinary wave (blue bar) progresses through the lower portion of the crystal at a higher velocity than does the extraordinary wave (red bar). When the waves encounter the cemented surface residing in the interior of the Wollaston prism, they under go angular wave splitting and each wave takes a slightly different course due to refraction at the interface. In addition, the orientation of the Wollaston prism crystalline axes reverses the refractive index differences at the boundary, resulting in the ordinary wave becoming the extraordinary wave as it enters the upper portion of the prism. A similar situation occurs with the extraordinary wave, which reverses roles to become the ordinary wave. This concept is demonstrated in the tutorial by changes in the wavefront bar color as each wave encounters the cemented prism junction (the blue wave becomes the red wave and vice versa).

When the tutorial initializes, the incident polarized light wave enters the lower central portion of the Wollaston prism and is diverted into an ordinary and extraordinary wavefront. As the wave travels through the lower portion of the prism, the ordinary wavefront (blue bar) progresses toward the cemented boundary faster than the extraordinary wavefront (red bar). After passing through the boundary and reversing identities, the extraordinary wavefront (which was originally the ordinary wavefront) slows down while the ordinary wavefront (originally the extraordinary wavefront) gains velocity. Eventually, the ordinary wavefront advances to an identical position with the extraordinary wavefront and the two emerge from the upper surface of the Wollaston prism simultaneously with zero path difference. Because each wavefront encounters an identical refractive index (that of the air) upon exiting the prism, the two waves travel at identical velocities on their way to the specimen.

In order to modify the optical path difference between the sheared waves, the Wollaston prism can be shifted in a direction perpendicular to that of the incident polarized light beam using the Prism Position slider. When the slider is translated to the left, the Wollaston prism also shifts to the left, decreasing the distance traveled through the lower portion of the prism by the wavefronts. In this case, the ordinary wavefront does not advance to a significant degree over the extraordinary wavefront before the cemented boundary is encountered. Upon reversal of the wavefront identities in the upper portion of the prism, the new ordinary wavefront progresses faster than the extraordinary wavefront and exits the prism surface first. This creates an optical path difference that can be varied by adjusting the position of the Wollaston prism. Alternatively, when the prism is shifted to the right by the slider, the ordinary wavefront advances to a considerable degree with respect to the extraordinary wavefront in the lower portion of the prism. When the boundary is encountered, there is only a short distance for each wave to travel before exiting the prism. In this case, the new extraordinary wavefront (previously the ordinary wavefront in the lower prism section) is far ahead of the ordinary wavefront and encounters the upper surface of the Wollaston prism first. The net result is an optical path difference that is opposite of the one demonstrated when the Wollaston prism is shifted to the left.

The Nomarski prism, like a Wollaston prism, consists of two optical quartz wedges cemented together at the hypotenuse. One of the wedges is identical to a conventional Wollaston quartz wedge and has the optical axis oriented parallel to the surface of the prism. However, the second wedge is modified by cutting the quartz crystal in such a manner that the optical axis is oriented obliquely with respect to the flat surface of the prism. When the wedges are combined to form a birefringent compound prism, the focal plane (and interference fringes produced when polarized light passes through the prism) lies outside the prism plate, as described above and illustrated in Figure 1. This effect occurs because shear now takes place at the air-quartz interface (Figure 1(b)), and refraction at the interface between the quartz wedges causes the sheared wavefronts to converge with a crossover point outside the prism. The actual position of the Nomarski prism focal plane can be adjusted over a range of several millimeters by altering the oblique angle of the optical axis in the second quartz wedge utilized to construct the prism (using the Prism Type slider in the tutorial).

Although Nomarski prisms are widely employed as objective prisms in modern differential interference contrast microscopes, there are fewer spatial constraints for condenser prisms, which can often be positioned precisely within the aperture plane. Therefore, a conventional Wollaston prism can sometimes be inserted into the microscope condenser, but in many cases, a Nomarski prism is used instead. When a Nomarski prism is utilized in the condenser, the prism is designed to produce an interference plane that is located much closer to the prism than those constructed for use with objectives. As a result, aside from being mounted in frames having different geometries, the two Nomarski prisms found in modern DIC microscopes are cut differently and are not interchangeable. In summary, for differential interference contrast microscopy, the condenser prism (also referred to as a secondary, auxiliary, or compensating compound prism) acts as a primary beamsplitter to shear the polarized wavefront, while the objective prism (the principal prism) recombines the separated waves and regulates the degree of retardation between the ordinary and extraordinary wavefronts.

The degree of shear for a particular Wollaston or Nomarski prism is set by the manufacturer and must coincide with that of a matching beam combining prism, located at the (effective) objective rear focal plane. Each sheared pair of light rays will pass through the condenser and be refracted by the lens elements so that the two beams travel parallel to each other as they leave the condenser and pass through the specimen. The beams are actually separated by a very small distance that is beneath the resolving power of the objective (the beam separation or shear distance, which usually ranges between 0.15 and 0.6 micrometers, is greatly exaggerated in the tutorial).

Because the individual light beams are derived from the same source prior to being sheared by a Wollaston or Nomarski prism, they are coherent and capable of interference. After leaving the condenser, the sheared light beams pass through closely adjacent regions of the specimen, which often induces an optical path difference between the two beams due to localized refractive index and thickness variations. Light beams exiting the specimen are captured by the objective and brought into focus at the rear focal plane, where a second Wollaston or Nomarski prism is strategically placed to recombine the beams into a common path. The paired light beams, still polarized and oriented with vibration directions that are perpendicular, next pass through a second polarizer (the analyzer). The analyzer has a polarization plane that is crossed with respect to the first polarizer and is oriented at a 45-degree angle to the beams exiting the second prism. Components of the light beams vibrating in the polarization plane of the analyzer are able to recombine and interfere to form the image observed in the microscope eyepieces or captured by a traditional or CCD camera system.

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., 110 Commerce Drive, 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.

Matthew J. Parry-Hill, Robert T. Sutter, and Michael W. Davidson - National High Magnetic Field Laboratory, 1800 East Paul Dirac Dr., The Florida State University, Tallahassee, Florida, 32310.

Differential Interference Contrast

Interactive Tutorials

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.

The tutorial initializes with a typical DIC microscope optical train equipped with Wollaston prisms appearing in the window. A beam of linearly polarized light emerges from the polarizer and enters the lower Wollaston prism (labeled the Condenser Prism) before being separated into two orthogonal components and sheared at the quartz wedge interface. In the tutorial, the ordinary wavefront is represented by short red lines (parallel to the browser window) on a black ray trace, while the extraordinary wavefront is depicted by black dots (perpendicular to the browser window) on a red ray trace. The sheared orthogonal wavefronts are rendered parallel by the condenser and pass through the specimen before being collected by the objective and focused onto the interference plane of the second Wollaston prism (the Objective Prism). Recombined wavefronts emerge from the objective prism and pass through the analyzer to complete their journey through the virtual DIC microscope.

In order to operate the tutorial, use the Objective Prism Position slider to translate the objective prism laterally across the optical path of the microscope. As the prism is moved to the left or right, the Emerging Waveform (depicted in the upper right-hand corner of the tutorial window) changes from linear, through varying degrees of elliptical, and finally, to circular. When the waveform has some degree of elliptical or circular character, a portion of the light emerging from the objective prism can then pass through the analyzer to form an image. The Specimen Gradient slider can be utilized to increase the optical path gradient across the specimen, which will also produce a change in the waveform emerging from the objective prism. Wavefront relationships in both Nomarski and Wollaston prisms can be examined by toggling between the two prism types using the Objective Prism radio buttons (labeled Wollaston and Nomarski). The speed of wavefronts traveling through the virtual microscope can be increased or decreased using the Applet Speed slider.

After emerging from the condenser Wollaston or Nomarski prism at the aperture plane, the sheared ordinary and extraordinary coherent wavefronts are focused by the lens elements of the condenser and travel through the specimen before being collected by the objective. Along their trajectories between the condenser and objective, the wavefronts remain parallel to one another and are separated by a shear distance derived from the geometrical constraints of the condenser prism. The spatial separation between wavefronts (shear distance) varies with condenser and objective numerical aperture, but has practical limits 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. Resolution in differential interference contrast can be increased (at the expense of contrast) by reducing the shear distance to about one-half the maximum resolution of the objective.

Most microscope manufacturers compromise on the shear distance versus resolution (and contrast) trade-off, and produce prisms that have a maximum shear distance of about 0.6 micrometers for lower magnification objectives (10x) down to a minimum approaching 0.15 micrometers for the higher magnification objectives (60x and 100x). Regardless of the shear distance, however, it is important to note that closely spaced wavefront pairs, spatially distributed across the entire microscope aperture, sample every point in the specimen to ultimately provide dual-beam interference at the image plane.

When undisturbed by the presence of a specimen, the coherent wavefront pairs experience identical optical path lengths between the specimen and image planes and arrive at the objective rear focal plane having the same phase relationship as when they left the condenser. The Nomarski prism located behind the objective recombines the wavefronts in the objective focal plane to generate linearly polarized light having an electric vector vibration orientation identical to that of the substage polarizer transmission axis. Linearly polarized wavefronts exiting the objective prism are blocked by the second polarizer (or analyzer), which has a transmission axis oriented perpendicular to that of the polarizer (Figure 1(a) and 1(b)). As a result, the image background observed in the viewfield appears very dark or black, a condition referred to as extinction.

Without specimen-induced phase shifts, the beamsplitting action of the condenser prism is exactly matched and reversed by the beam-recombining effect of the objective Nomarski prism to ultimately produce linearly-polarized light. In other words, when the microscope is properly configured for Köhler illumination (a critical prerequisite for high-resolution differential interference contrast microscopy), the condenser and objective operate in conjunction to project an image of the light source and condenser prism onto the objective prism. The objective Nomarski prism, which is inverted in orientation with respect to the condenser prism, introduces a phase shift that exactly compensates the linear phase shift between the wavefronts produced by the condenser prism. This action occurs for all paired wavefronts across the entire microscope aperture. The axes of both the condenser and objective prisms are parallel to each other and oriented at a 45-degree angle with respect to the transmission axes of the crossed polarizers (polarizer and analyzer). The orientation axis of the two prisms is termed the shear axis, an important concept that defines the axis of lateral separation between the ordinary and extraordinary wavefronts from the time they leave the condenser prism until they are recombined by the objective prism and arrive at the image plane.

In the event that coherent paired wavefronts encounter a phase gradient present in the specimen while passing from the condenser to the objective, wavefront distortion is induced and the waves will undergo a phase shift along the shear axis and traverse slightly different optical paths (although no change in polarization occurs). Upon arriving at the objective prism, the phase-shifted paired wavefronts are recombined to generate elliptically polarized light (in contrast to the linearly polarized light produced in the absence of a specimen). The electric vector of the resultant wavefront, which is no longer planar, sweeps out an elliptical pathway as it travels through the region between the objective prism and the analyzer (as illustrated in Figure 1(c)). Because a component of the elliptical wavefront is now parallel to the transmission axis of the analyzer, some portion of the wave will pass through the analyzer and produce plane-polarized light having a finite amplitude and ultimately being able to generate intensity in the image plane.

In summary, 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 or 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, but simple, darkfield illumination technique.

Contributing Authors

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.

Jan Hinsch - Leica Microsystems, Inc., 110 Commerce Drive, Allendale, New Jersey, 07401.

Matthew J. Parry-Hill, Robert T. Sutter, and Michael W. Davidson - National High Magnetic Field Laboratory, 1800 East Paul Dirac Dr., The Florida State University, Tallahassee, Florida, 32310.

Differential Interference Contrast

Interactive Tutorials

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.

The tutorial initializes with a randomly selected DIC image appearing in the Specimen Image window having a small bias retardation value (one-twentieth of a wavelength) applied to the optical system. To operate the tutorial, translate the Bias Retardation slider to higher values (to the right) in order to produce higher-order interference colors in the specimen image. This slider has an operational range between 1/20 and a full wavelength of retardation. New specimens can be examined by selecting the appropriate choice in the Choose A Specimen pull-down menu.

Bias retardation between the ordinary and extraordinary wavefronts in differential interference contrast can be manipulated through the use of compensators originally targeted as quantitative retardation measuring devices and contrast-enhancing elements for polarized light microscopy. Compensating plates bestow greater control for adjusting the contrast of specimen details in relation to the background intensity and color values, and also enable more precise tuning of the bias value between wavefronts. These birefringent components are also frequently employed for optical staining of transparent specimens, which are normally rendered over a limited range of grayscale values.

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.

Compensators can be inserted into the optical pathway of a DIC microscope between either the objective prism and the analyzer or the polarizer and the condenser prism. Many microscopes have a slot located in the intermediate tube or substage condenser housing designed for this purpose. Addition of a first-order compensator (often termed a full-wave or first-order red plate) having a retardation value equal to a full wavelength in the green region of visible light (approximately 550 nanometers), introduces a spectrum of interference colors to the specimen and background. With the compensator in place, green light is unable to pass through the analyzer because it emerges from the retardation plate linearly polarized with an electric field vector having the same orientation as the polarizer. However, wavefronts in the red and blue spectral regions experience retardations less than a wavelength and become elliptically polarized, allowing them to pass a component through the analyzer. As a result, these colors become mixed to form a magenta background in the field of view.

Thus, when a specimen is observed in white light with differential interference contrast optics and a first-order compensator, the background appears magenta while image contrast is displayed in the second-order blue and first-order yellow colors (depending on orientation) of the Newtonian interference color spectrum. With the compensator in place, small variations in bias retardation obtained by translation of the Nomarski prism (or rotating the polarizer in a de Sénarmont compensator) yield rapid changes to interference colors observed in structures having large path length gradients. This technique is useful for introducing color (optical staining) to regions having high refractive index boundaries, such as cellular membranes, large intracellular particles, cilia, and the nucleus. The interference colors displayed by specimen features can be compared to the values on a Michel-Levy color chart to obtain an estimate of the optical path difference.

Illustrated in Figure 1 are several transparent specimens that have been optically stained and rendered in pseudo three-dimensional relief through DIC optical techniques. Figure 1(a) depicts projections at the edge of a ctenoid fish scale, while the mouth of a canine hookworm (Ancylostoma caninum) is featured in Figure 1(b). Colorful wing scales of the Great Leopard moth (Ecpantheria scribonia) are presented in Figure 1(c). In all cases, the Nomarski prism was translated across the microscope optical axis to a bias retardation value exceeding a full wavelength. Although these images do not reveal hidden scientific information pertaining to the specimens, they do have the potential to advance the technique of DIC optical microscopy as a legitimate bridge between science and art.

On microscopes equipped with a de Sénarmont compensator for introducing bias into a differential interference contrast optical system, a full-wave retardation plate can be added to optically stain the specimen with Newtonian interference colors and provide more quantitative information about path differences. The de Sénarmont compensator is frequently employed in DIC microscopy to obtain precisely measured levels of bias retardation, but the device is also useful to monitor alignment of the optical components. In video-enhanced DIC (VE-DIC) microscopy, de Sénarmont compensators are often utilized to optimize contrast in specimen detail that lies below the resolution limit of the microscope.

Contributing Authors

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.

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

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

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

Differential Interference Contrast

Interactive Tutorials

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 lateral 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 that differ in thickness.

The tutorial initializes with a randomly selected image appearing in the DIC Specimen Image window and a model of an objective and specimen on the right-hand side of the window. In order to operate the tutorial, use the Objective Focus slider to focus the specimen through a range of planes starting at the upper surface and progressing through the central region to the lower surface. As the slider is translated, various image planes in the specimen will be brought into sharp focus. Simultaneously, the microscope objective model will move upward and downward with respect to the specimen to simulate the actual relationship in the microscope. A new specimen can be examined by selecting from the palette in the Choose A Specimen pull-down menu.

In all traditional forms of transmitted and reflected light microscopy, the condenser aperture iris diaphragm plays a major role in defining image contrast and resolution. Reducing the aperture size increases the depth of field and overall image sharpness while simultaneously producing enhanced contrast. However, if the diaphragm is closed too far, diffraction artifacts become apparent and resolution is sacrificed. Often, the optimum aperture setting is a compromise between accurately rendering specimen detail in sufficient contrast and retaining the resolution necessary to image minute features while avoiding diffraction artifacts.

High-performance differential interference contrast microscope optical systems produce excellent contrast with the condenser iris diaphragm partially closed (approximately 70 percent of the objective rear aperture size), but also perform superbly when the diaphragm is opened to match the objective aperture size. In order to achieve the optimum balance between resolution and contrast for optical sectioning, the microscope must be properly configured for Köhler illumination and the prism components and polarizers should be accurately aligned. High numerical aperture objectives designed for oil immersion should only be utilized to image specimen slides that have the underside oiled to the condenser.

At high magnifications and numerical apertures (for example, 100x and 1.4), the depth of field in differential interference contrast approaches a limiting value of about 400 nanometers (0.40 micrometers; about 1.5 times the lateral resolution limit of the objective). Optical sections taken through the central region of a human buccal epithelial cell at high magnification (100x objective) and numerical aperture (1.30) are illustrated in Figure 1. The cell is approximately 3 micrometers thick near the nucleus, and depth of field for the configuration utilized to obtain the images in Figure 1 was approximately 0.5 micrometers. Bacteria are clearly visible on the upper surface of the cell (Figure 1(a)), as are parallel, swirling ridges in the membrane that closely resemble the texture of human fingerprints. A bulge in the membrane corresponding to the nucleus is also visible in the lower central portion of the figure. As the focal plane is shifted to the interior of the cell (Figure 1(b)), details of the nuclear structure and intracellular particles become visible. Finally, at the lower boundary where the cellular membrane rests on the surface of the microscope slide (Figure 1(c)), numerous folded ridges are revealed (similar to those observed on the upper surface).

When performing optical sectioning experiments with thicker biological specimens (especially those immersed in aqueous saline solutions), the microscopist must be alert to the possible introduction of spherical aberration produced by refractive index discontinuities at the interface between the coverslip and mounting medium. These artifacts will reduce resolution at higher penetration depths in the optical section series.

A significant amount of the current research and development of theory in differential interference contrast is focused on the striking optical sectioning characteristics of the technique. Ultimately, a quantitative estimate of the three-dimensional refractive index profile from a specimen may be achieved through computational models. In addition, current research is also directed at new models being developed for the DIC optical components and image formation in partially coherent transmitted light.

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.

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

Jan Hinsch - Leica Microsystems, Inc., 110 Commerce Drive, Allendale, New Jersey, 07401.

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

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.

Figure 1 - DIC Microscope Configuration with de Sénarmont Bias Retardation Control

The basic de Sénarmont configuration for a modern, upright research instrument is illustrated in Figure 1 for the Nikon Eclipse E600 microscope series. The polarizer and quarter-wavelength retardation plate are combined into a housing that is secured with a setscrew onto the main light port in the base of the microscope. Once fixed into position, the retardation plate is held in a preset orientation, while the polarizer can be rotated through 90 degrees (plus or minus 45 degrees) around the optical axis of the microscope. Depending upon the orientation of the polarizer with respect to the retardation plate, the de Sénarmont compensator will illuminate the microscope optical train with either linear, elliptical, or circularly polarized light.

Polarized wavefronts exiting the de Sénarmont compensator first encounter the fixed Nomarski prism in the microscope condenser turret (see Figure 1), and are subsequently re-oriented and sheared to produce orthogonal components (an ordinary and an extraordinary wavefront) having vibration directions at a 45-degree angle to the polarized light emerging from the de Sénarmont compensator. The condenser lens system focuses the sheared wavefronts into parallel components and projects them onto the specimen. Light leaving the specimen is collected by the objective and focused onto the interference plane of the second fixed Nomarski prism located in a frame housed in the microscope nosepiece. The objective Nomarski prism, which is matched to the condenser prism and inverted, recombines the sheared wavefronts into coaxial orthogonal components. Although linearly polarized light exiting the objective Nomarski prism is blocked by the analyzer, elliptical and circularly polarized light can pass components through to form an image of the specimen.

Differential interference contrast microscopy was first suggested and devised by Francis Smith in 1955, who built a modified polarized light microscope having Wollaston prisms added to the front focal plane of the condenser and the rear focal plane of the objective. Because of their design limitations, Wollaston prisms were subsequently replaced with a more advanced system, introduced by French scientist Georges Nomarski, which enabled the prisms to be physically located some distance away from the condenser and objective aperture conjugate planes. This modification allows the use of standard microscope optical components in modern DIC systems, and is currently enjoying widespread application.

In a manner similar to phase contrast, DIC microscopy is useful for visualizing living cells and other transparent, unstained specimens that are otherwise difficult to observe in traditional brightfield illumination using the full aperture and resolution of the microscope optical system. However, DIC does not suffer from the halo artifacts and masked apertures inherent with phase contrast optics, and has the ability to produce excellent images with relatively thick specimens. In addition, DIC images can be easily manipulated using digital and video imaging techniques to further enhance contrast.

Figure 2 - Origin of Contrast in DIC Microscopy

The differential interference contrast effect functions by converting gradients in specimen optical path length into amplitude (intensity, as illustrated in Figure 2) differences that can be visualized as improved contrast in the microscope eyepieces (or recorded on film or digitally). The primary determining factors in specimen optical path length are the refractive index difference between the specimen and its surrounding medium, and the geometrical distance traversed by the sheared wavefronts between two points on the optical path. Images produced by DIC optical systems have a distinctive shadow-cast appearance and appear to be pseudo three-dimensional, as if they were illuminated from a highly oblique light source originating from a single azimuth. In general, DIC is useful for determining the orientation of phase gradients and for utilizing the full objective aperture to produce thin optical sections of specimens that are devoid of obscuring disturbances positioned beyond the immediate focal plane.

Wollaston and Nomarski Prisms

The critical elements in differential interference contrast microscopy are matched birefringent Wollaston and/or Nomarski prisms, which are inserted into the microscope optical pathway with their shear axes oriented at a 45-degree angle (northwest to southeast) to the polarizer and analyzer. Wollaston prisms are constructed of two precisely ground and polished wedge-shaped slabs of quartz having perpendicular orientations of the optical axis and cemented together at the hypotenuse to generate an optically anisotropic compound plate. Incident linearly polarized wavefronts entering a Wollaston prism (oriented at a 45-degree angle to the prism optical axes) are divided into two separate orthogonal waves, termed the ordinary and the extraordinary wave.

Because they are derived from localized areas on a single source (generally, the filament of a tungsten-halogen lamp), the orthogonal wavefronts generated by a Wollaston prism are coherent, have identical amplitudes, and propagate in the same direction through the lower prism wedge (see Figure 3(a)). The ordinary wave travels through the prism in a direction that is perpendicular to the optical axis, while the extraordinary wave is oriented parallel to the axis. In addition, varying dielectric properties along these perpendicular axes result in one wave traveling through the prism at a higher velocity than the other. The ordinary wave proceeds through the prism along the faster axis (having a lower refractive index), and the extraordinary wave traverses the slower axis (higher refractive index).

Figure 3 - Wollaston and Nomarski Prisms for DIC Microscopy

At the refractive index junction between the cemented quartz wedges, an angular splitting or shear of the wavefronts occurs, resulting in the waves becoming spatially separated by an angle defined as the shear angle. In addition, the wavefronts exchange identities at the prism wedge boundary, with the ordinary wave becoming the extraordinary wave and vice versa. The shear angle and separation distance is constant for all incident wavefronts across the prism, regardless of the entry point, and the direction of wavefront shear is defined by the prism shear axis. This axis lies in the longitudinal plane of a Wollaston prism and is parallel to the direction of the optical axis of the lower quartz wedge. In general, the shear distance produced by a Wollaston (or Nomarski) prism is less than a micrometer so that no observable beam separation occurs in light emerging from the prism.

The refractive index difference experienced by the ordinary and extraordinary wavefronts results in varying propagation velocities for the two waves as they travel through the lower and upper sections of a Wollaston prism. In the lower half of the prism, the wavefronts undergo a phase shift that is exactly compensated in the upper half when the geometrical path through the lower and upper halves is identical. However, wavefronts traversing through points away from the center experience a longer journey through either the lower or upper half of the prism, which results in a constant phase shift per unit length in the direction of the shear axis. The phase shift is equal, but opposite, for the ordinary and extraordinary wavefronts. As a result, at one end of the prism, the extraordinary wavefront emerges ahead of the ordinary wavefront, while at the corresponding position on the other end, the ordinary wavefront exits the prism before the extraordinary wavefront. The focal, or interference, plane of a Wollaston prism occurs in the center of the prism where the optical path between the lower and upper quartz wedges is equal. For this reason, it is often difficult (or impossible) to adapt a Wollaston prism for use with standard microscope objectives, which often have a rear focal plane (that must physically coincide with the Wollaston prism interference plane) buried deep inside a lens.

Similar to a conventional Wollaston prism, the basic Nomarski prism design (Figure 3(b)) also consists of two quartz wedges that are cemented together at the hypotenuse. The upper wedge is identical to a conventional Wollaston quartz wedge, but the lower wedge is modified by cutting the quartz crystal in a direction that yields an oblique optical axis with respect to the flat surface of the prism. When the wedges are combined to form a compound birefringent prism, the focal (interference) plane lies at a site several millimeters outside the prism plate. This effect occurs because shear takes place at the quartz-air interface in the lower prism wedge instead at the cemented boundary, as in a Wollaston prism. Refraction at the interface between the quartz wedges in a Nomarski prism causes the sheared wavefronts to converge with a crossover point outside the prism (as illustrated in Figure 3(b)). The actual position of the Nomarski prism focal plane can be adjusted over a range of several millimeters by altering the oblique angle of the optical axis in the lower quartz wedge utilized to construct the prism.

Modern DIC microscopes are usually constructed with Nomarski prisms to avoid the objective aperture clearance problem. In fact, the modified prisms are often employed for both beam shearing and recombination duty in the condenser and objective focal planes. Although there are fewer spatial constraints for condenser prisms, which can often be positioned precisely within the aperture plane, a Nomarski prism is still used in most cases. Nomarski prisms can be designed specifically for condensers to produce an interference plane that is located much closer to the prism than those constructed for use with objectives. As a result, aside from being mounted in frames having different geometries, the two Nomarski prisms found in modern DIC microscopes are often cut differently and are not interchangeable.

The de Sénarmont DIC Optical System

A traditional differential interference microscope optical system contains a polarizer located before the condenser and an analyzer (a second polarizer) inserted into the pathway above the objective, usually in an intermediate tube or combined in the frame with the objective Nomarski prism. The polarizer is oriented with the vibration plane transmission axis positioned East-West, while the analyzer is crossed with respect to the polarizer (transmission axis is North-South). Linear polarized light leaving the polarizer is separated into two components by a Nomarski prism housed in the condenser near the conjugate focal plane of the iris diaphragm aperture. Incident wavefronts are sheared by the prism into orthogonal polarized components, rendered parallel by the condenser optical system, and then utilized to illuminate the specimen.

Positioned behind the objective in the optical pathway is a second Nomarski prism (usually housed in an adjustable sliding frame), which is utilized to recombine the sheared wavefronts in the conjugate plane of the rear aperture after they have been collected and focused by the objective. Components of circular and elliptically polarized light from the recombined wavefronts pass through the analyzer and subsequently undergo interference to generate the DIC image at the microscope intermediate image plane.

Figure 4 - de Sénarmont Compensator Wavefronts

In a perfectly aligned DIC microscope, the condenser prism is imaged by the condenser and objective lens assemblies onto the objective prism so that wavefront shear is matched at every point along the surface of the prisms, which are inverted with respect to one another. Translating either prism along the shear axis (perpendicular to the microscope optical axis) produces a wavefront mismatch that is uniform across the microscope aperture. Shifting the phase displacement of the ordinary wavefront with respect to the extraordinary wavefront through translation of a prism is often termed introduction of bias retardation in DIC microscopy. As one of the Nomarski prisms is shifted laterally (usually the objective prism), wavefront pairs contributing to the background become increasingly retarded and out of phase with one another. Thus, the polarization vector orientation of light emerging from the objective Nomarski prism can be adjusted from linear (no translation), through varying degrees of elliptical, and even to circular.

Introduction of bias retardation into a DIC optical system produces changes to the intensity (amplitude) levels of phase gradients in the specimen, which result in the generation of orientational-dependent bright highlights and dark shadows superimposed on a lighter background. Intensity gradients occur along the shear axis of the condenser and objective prisms, and generally appear to be originating from a 45-degree angle (northwest to southeast or vice versa) when the specimen is observed in the eyepieces. Shifting the prism in one direction or another across the microscope optical axis will vary the phase relationship between the orthogonal wavefronts, thus reversing the shadow-cast orientation in the specimen. The net result is to render 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.

An alternative technique for introduction of bias retardation, which is growing in popularity, is to mount a quarter-wavelength retardation plate in fixed orientation between the polarizer and condenser prism (termed de Sénarmont DIC compensation, as discussed above). At maximum extinction (matched prisms with no bias applied; see Figure 4(b)), the fast axis of the retardation plate is aligned with the transmission axis of the polarizer, and both optical units can be (and often are) contained within the same housing on the base of the microscope. An alternative location for the de Sénarmont compensator, in microscopes equipped with the appropriate intermediate tube (see Figure 1), is between the objective prism and the analyzer.

In order to introduce bias using the de Sénarmont compensator, the polarizer transmission axis is rotated (up to plus or minus 45 degrees; see Figures 4(a) and 4(c)) with respect to the fast axis of the retardation plate, which remains fixed at a 90-degree angle relative to the analyzer transmission axis. When the compensator fast axis coincides (is parallel) with the transmission axis of the polarizer, only linearly polarized light passes through the de Sénarmont compensator to the condenser prism, as illustrated in Figure 4(b). However, when the polarizer transmission axis is rotated, wavefronts emerging from the quarter-wavelength retardation plate become elliptically polarized. Rotating the polarizer in one direction will produce right-handed elliptically polarized light (Figure 4(a)), while rotating the polarizer in the other direction will alter the vector trajectory to generate a left-handed elliptical sweep (illustrated in Figure 4(c)).

Figure 5 - Bias Retardation in de Sénarmont DIC Microscopy

When the orientation of the polarizer transmission axis reaches either plus or minus 45 degrees (equivalent to one-quarter wavelength of retardation), light passing through the compensator becomes circularly polarized (again in either a left-handed or right-handed sense). Because elliptically or circularly polarized light represents a phase difference between the ordinary and extraordinary wavefronts emerging from the de Sénarmont compensator, bias is introduced to the system when the wavefronts enter the condenser Nomarski beamsplitter prism and become sheared (Figure 5). Positive bias is obtained when the polarizer is rotated in one direction, while negative bias is introduced by rotating the polarizer in the opposite direction. The amount of retardation introduced by a de Sénarmont compensator can be quantitatively determined according to the equation:

Retardation (in nanometers) = θλ/180

where θ is the rotation angle (calculated in degrees) of the polarizer transmission axis in relation to the fast axis of the retardation plate, and λ is the average wavelength of light passing through the compensator. In the case of tungsten-halogen illumination, the wavelength is often taken to be approximately 550 nanometers for calculations involving bias retardation. With de Sénarmont compensators, bias retardation values ranging between one-twentieth and a full wavelength can be easily measured with an accuracy of 0.15 nanometers.

The effects of bias retardation introduced into the DIC optical system by a de Sénarmont compensator are illustrated in Figure 5 for three settings of the compensator. All of the examples presented in Figure 5 diagram a single wavefront entering the central portion of a Wollaston prism, but a Nomarski prism will operate in the same fashion. When the polarizer transmission axis is aligned parallel to the fast axis of the quarter-wavelength retardation plate (Figure 4(b) and 5(b)), linearly polarized light emerges from the compensator and is incident on the lower wedge surface of the condenser Wollaston prism (as depicted in Figure 5(b)). In a DIC microscope, the incident linearly polarized light is oriented at a 45-degree angle to the fast and slow axes of the lower wedge component of the Wollaston (or Nomarski) prism. Upon entering the prism, the polarized light is separated into orthogonal components, which traverse the fast and slow axes of the lower quartz wedge and become sheared at the boundary between the two prism wedges. Because the linear wavefront exists as a single phase when it enters the prism, the orthogonal components are initially in phase when they are produced at the air-quartz boundary, but undergo phase shifts inside the prism.

As previously described, the gain in phase by the ordinary wavefront in the lower portion of the Wollaston prism is offset in the upper wedge after the ordinary and extraordinary waves exchange identities at the junction between the two wedges. The net result is a cancellation of phase shifts produced inside the prism, and the orthogonal wavefronts emerge from the Wollaston prism in phase with each other (Figure 5(b)). In this condition, the optical system exhibits maximum extinction, and only large phase gradients present in the specimen are visible. These phase gradients are superimposed on a very dark, or black, background and take on the appearance of a darkfield image.

The situation is quite different when the polarizer is rotated in either direction away from the zero position in a de Sénarmont compensator. Wavefronts emerging from the compensator exhibit phase shifts that impart an elliptical or circular character to the vector summation of the orthogonal wave components. When the polarizer is rotated to the left by 30 degrees (negative bias retardation; Figure 5(a)), the ordinary wavefront generated by the de Sénarmont compensator enters the Wollaston prism prior to the extraordinary wavefront, and exits the prism (after exchanging identities) as the extraordinary wavefront ahead of the ordinary wavefront. The basic result is the generation of an optical path difference, which is required for the introduction of bias retardation. The opposite effect occurs (Figure 5(c)) when the de Sénarmont polarizer is rotated to the right (positive bias retardation), and the ordinary wavefront emerges from the Wollaston prism ahead of the extraordinary wavefront.

Regardless of whether bias is introduced into a differential interference contrast system by translating the objective Nomarski prism or by rotating the polarizer on a de Sénarmont compensator, the net result is the same. In a properly configured microscope that is correctly aligned for Köhler illumination, an image of the light source and condenser prism is transferred by the optical system (condenser and objective) onto the inverted second Nomarski prism located at the objective rear focal plane. The linear phase shift across the face of the condenser prism is precisely compensated by an opposite phase shift in the objective prism. Translation of the objective prism along the shear axis does not alter the phase shift distribution, but instead, adds or subtracts a constant phase difference across the entire microscope aperture. In the same manner, rotating the polarizer in a de Sénarmont compensator also introduces a variable and controlled phase difference. The matched prism system enables image formation to occur with the same bias retardation for every wavefront pair projected from the condenser aperture, irrespective of the route through which it traverses the specimen to reach the objective.

Figure 6 - Bias Retardation with de Sénarmont Compensation

Presented in Figure 6 is a series of digital images recorded in differential interference contrast using a bias retardation range of one-twentieth to a quarter wavelength in several intermediate steps. The specimen is a 5-micrometer (thickness) preparation of human buccal mucosa epithelial (cheek) cells spread on a microscope slide in buffered saline solution and imaged through a 170-micrometer cover glass. The cells exhibit regions of fluctuating thickness, which range from approximately 1 to 4 micrometers. Rendition of specimen detail and the shadow-cast pseudo three-dimensional effects are the most pronounced at the lower bias retardation values (Figure 6(a) and 6(b)), but contrast and definition of fine specimen detail both deteriorate as bias retardation is increased (Figure 6(c) through 6(f)). At the highest bias retardation value (one-quarter wavelength; Figure 6(f)), contrast is extremely poor and very few structural details are visible. For this particular specimen, the optimum retardation range appears to lie between one-twentieth and one-twelfth of a wavelength.

As the optical path gradient in a specimen increases in DIC microscopy, so does image contrast. Altering the bias retardation to varying degrees can also produce significant contrast fluctuations in the specimen as observed in the eyepieces (Figure 6). In general, the optimum degree of displacement between the ordinary and extraordinary wavefronts induced by translation of the objective prism, or by rotating the polarizer in a de Sénarmont compensator, is on the order of less than one-tenth wavelength. However, this value is largely dependent on specimen thickness, and the useful range of bias retardation for biological specimens lies between one-thirtieth and a quarter wavelength. Contrast in specimens having very large optical gradients can often benefit from even greater bias retardation values (up to a full wavelength). The introduction of bias retardation into a differential interference contrast microscope enables phase specimens to be observed with greater ease, and dramatically facilitates imaging efforts with traditional film or digital camera systems.

Compensator Retardation Plates in de Sénarmont DIC Microscopy

Commonly referred to as compensating or retardation plates in the field of polarized light microscopy, compensators can be utilized in DIC microscopy to manipulate the amount of contrast obtained from bias retardation between the ordinary and extraordinary wavefronts. Compensating plates bestow greater control for adjusting the contrast of specimen details in relation to the background intensity and color values, and also enable more precise tuning of the bias retardation between wavefronts. These birefringent components are also frequently employed for optical staining of transparent specimens, which are normally rendered over a limited range of grayscale values.

On microscopes equipped with a de Sénarmont compensator for introducing bias into a differential interference contrast optical system, a full-wave retardation plate can be added to optically stain the specimen with Newtonian interference colors and provide more quantitative information about path differences (illustrated in Figure 7). Retardation plates can be inserted into the optical pathway of a de Sénarmont DIC microscope between either the objective prism and the analyzer or the compensator and the condenser prism. Many microscopes have a slot located in the intermediate tube or substage condenser housing designed for this purpose. Addition of a first-order compensator (often termed a full-wave or first-order red retardation plate) having a retardation value equal to a full wavelength in the green region of visible light (approximately 550 nanometers), introduces a spectrum of interference colors to the specimen and background (Figure 7). With the compensator in place, green light is unable to pass through the analyzer because it emerges from the retardation plate linearly polarized with an electric field vector having the same orientation as the polarizer. However, wavefronts in the red and blue spectral regions experience retardations less than a wavelength and become elliptically polarized, enabling them to pass a component through the analyzer. As a result, these colors become mixed to form a magenta background in the field of view.

Figure 7 - Optical Staining with de Sénarmont DIC and a Full-Wave Plate

When a specimen is observed in white light (tungsten-halogen illumination) with a de Sénarmont DIC optical system and a first-order retardation plate, the background appears magenta while image contrast is displayed in the second-order blue and first-order yellow colors (depending upon orientation) of the Newtonian interference color spectrum. With the retardation plate inserted into the light pathway, small variations in bias retardation obtained by rotating the polarizer in a de Sénarmont compensator yield rapid changes to interference colors observed in structures having large optical path length gradients. This technique is useful for introducing color (optical staining) to regions having high refractive index boundaries, such as cellular membranes, large intracellular particles, cilia, and the nucleus. The interference colors displayed by specimen features can be compared to the values on a Michel-Levy color chart in order to obtain an estimate of the optical path difference.

Illustrated in Figure 7 are several semi-transparent specimens that have been imaged through both standard bias retardation techniques and optical staining, and rendered in pseudo three-dimensional relief with a vivid display of color. Figure 7(a) depicts striations in a ctenoid fish scale at a bias retardation setting of approximately one-twelfth of a wavelength. Inserting a first-order retardation plate into the microscope optical pathway produces the image illustrated in Figure 7(b), which renders the ctenoid scale in second-order blue and first-order yellow interference colors on a magenta background. Likewise, an Obelia hydroid polyp displays a significant amount of structure at low to intermediate bias retardation values (Figure 7(c)), but also exhibits an abundant spectrum of color with the retardation plate in the light path (Figure 7(d)). Optical staining can also be useful to produce color reversal of shadow-cast effects, as shown in Figure 7(e) and 7(f) for a transparent thick section of murine kidney tissue. Although the images presented in Figure 7 do not reveal hidden scientific information pertaining to the specimens, they do have the potential to advance the technique of DIC optical microscopy as a legitimate bridge between science and art.

Interpretation of de Sénarmont DIC Images

By observing the shadow-cast orientation present in almost every image produced through bias retardation introduced by a de Sénarmont compensator, the direction of optical shear is obvious and can be precisely defined as the axis connecting regions displaying the highest and lowest intensity (amplitude) values. The level of contrast bestowed on specimen phase gradients (and the degree of pseudo three-dimensionality) by de Sénarmont DIC is a function of the amount of bias retardation introduced into the optical system by rotating the polarizer in the compensator. Because the shear axis is fixed by Nomarski prism design, and other constraints involved in wavefront orientation for DIC microscopy, the axis direction cannot be altered to affect specimen contrast through a simple setting on the microscope. However, the relative phase retardation between ordinary and extraordinary wavefronts can be reversed by rotating the de Sénarmont compensator polarizer to the corresponding negative value. When the phase retardation is altered as just described, the orientation of bright and dark edges on the specimen is reversed by 180 degrees. In essence, the only mechanism available to the microscopist to change the shear axis relative to the specimen is to reorient the specimen itself, a maneuver that benefits from the utilization of circular 360-degree rotatable stages.

Each specimen examined in de Sénarmont DIC microscopy will have an optimum bias retardation setting of the compensator that generates a maximum level of contrast in the final image. Very thin specimens that display a shallow refractive index gradient, such as living cells in culture, generally benefit from equally low bias settings (small translations of the polarizer) only slightly larger than the largest phase shift present in the specimen (on the order of approximately one-twentieth of a wavelength, or about 30 nanometers). However, thicker specimens often require higher bias settings (up to a quarter-wavelength) at large condenser apertures to produce satisfactory results, usually through optical sectioning. Because many specimens are composed of features displaying a variety of different sizes and refractive indices, the optimum bias retard setting of the de Sénarmont compensator is usually a compromise.

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 optical sections from a focused image plane that are remarkably shallow. 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.

In all traditional forms of transmitted and reflected optical microscopy, the condenser aperture iris diaphragm plays a major role in defining image contrast and resolution. Reducing the aperture size increases the depth of field and overall image sharpness while simultaneously producing enhanced contrast. However, if the condenser diaphragm is closed too far, diffraction artifacts become apparent and resolution is sacrificed. Often, the optimum aperture diaphragm setting is a compromise between accurately rendering specimen detail in sufficient contrast and retaining the resolution necessary to image minute features while avoiding diffraction artifacts.

Figure 8 - Optical Sectioning in de Sénarmont DIC Microscopy

When the condenser iris diaphragm is adjusted to approximately 70 percent of the objective rear aperture size, most high-performance de Sénarmont DIC optical systems produce excellent contrast. However, these microscopes also perform superbly when the condenser diaphragm is opened to match the objective rear aperture diameter. In order to achieve the optimum balance between resolution and contrast for optical sectioning experiments, it is critical that the microscope be properly configured for Köhler illumination and the Nomarski prism components, analyzer, and de Sénarmont compensator be accurately aligned.

Optical sections taken of a living Volvox colony using de Sénarmont DIC to achieve bias retardation on an inverted tissue culture microscope are illustrated in Figure 8. The aquatic microorganism is composed of hundreds to thousands of identical green algae cells having approximately the same diameter, but organized into several morphological motifs. On the periphery of the colony, individual cells are arranged in a semi-transparent widely spaced layer termed the mucilage, as illustrated in Figure 8(a). Farther into the mass, the colony forms several concentrated spherical groups of reproductive cells, termed gonidia, which produce small daughter colonies within the parent colony (Figure 8(b)). As the microscope is focused on the uppermost layer of the colonies in the mucilage (Figure 8(c)), structural detail in the individual cells becomes visible, but many cells are masked by the daughter colony.

Thin biological specimens (10 to 20 micrometers in thickness) usually produce poor optical sections at lower magnifications, but often reveal substantial internal detail when visualized with high magnification objectives having large numerical aperture (60x and 100x). Thicker specimens can be readily sectioned at low magnifications where aberration is minimized. Collection of optical sections from thicker biological specimens, especially those immersed in aqueous saline or buffered solutions, is often hampered by spherical aberration produced by refractive index discontinuities at the interface between the cover glass and mounting medium. This artifact will reduce resolution at higher penetration depths in the optical section series.

Conclusions

In differential interference contrast microscopy, a phase difference is introduced by geometric path length and refractive index variations of the specimen into the two orthogonal wavefronts generated by the Nomarski prism housed in the condenser. The net result is elliptical polarization between the wavefronts that are recombined by the objective Nomarski prism and subsequent image formation as a component of these waves passes through the analyzer. Bias retardation can be introduced to the system by translating the objective prism along the microscope optical axis or by combining a quarter-wavelength plate with the polarizer or analyzer (de Sénarmont compensation). Thus, optimum contrast, field brightness, and sensitivity can be gained by simple rotation of a control knob. There are several advantages to using de Sénarmont compensation DIC microscopy instead of the traditional configurations that rely on translation of the objective Nomarski prism, and these benefits are partially responsible for the increasing popularity of the technique.

A quantitative determination of the exact amount of bias retardation applied to the optical system is difficult to ascertain with a majority of DIC microscopes that employ a translating Nomarski prism in the objective rear focal plane. In some cases, Nomarski prism frames with highly accurate micrometer-controlled translation devices have been developed, and these components can be utilized to accurately gauge the amount of bias introduced by moving the prism in small increments. However, any DIC microscope can be configured for accurate bias retardation measurements using de Sénarmont compensation simply by adding a graduated rotating polarizer or analyzer and a fixed quarter-wavelength retardation plate to the optical train.

One of the most convenient configurations for quantitative bias retardation measurement is illustrated by the microscope presented in Figure 1. This instrument contains an intermediate tube on the main frame positioned between the objective Nomarski prism in the nosepiece and the observation tubes. Inserted into the intermediate tube is a standard 550 nanometer de Sénarmont compensator (originally designed for polarized light measurements) housed in a fixed frame with the fast axis parallel to the polarizer (East-West) and the slow axis parallel to the analyzer (North-South). Above the de Sénarmont compensator is a 360-degree adjustable frame containing a linear polarizing element (acting as the analyzer), and graduated in single degrees. The circular control knob often is accompanied by a vernier scale that enables accurate determination of the linear polarizer transmission axis orientation and provides the microscopist with the ability to control (and gauge) the level of bias retardation to a few fractions of a wavelength. When a DIC microscope originally designed to introduce bias retardation by translation of the objective Nomarski prism is configured as just described, accurate determination of bias retardation is possible. Under these conditions, however, it is important to initially configure the matched prisms (objective and condenser) for maximum overlap of the interference fringes (maximum extinction) prior to inserting the de Sénarmont compensator and rotating analyzer.

Another important advantage of de Sénarmont DIC compensation is the proximity of these components to important image forming and aperture conjugate planes in the microscope optical train. Microscopes designed with sliding objective Nomarski prisms must physically translate the compound prism element across the optical pathway while the microscopist observes the level of bias retardation applied to the system. In many cases, especially if the prism frame fits loosely in the mount, serious image shifts can occur during prism translation, which impede the ability to observe the specimen and to capture images. Image shifts cannot occur during introduction of bias retardation with a de Sénarmont DIC microscope, primarily because the compensator retardation plate and rotating polarizer are positioned before the condenser or after the objective, in the microscope illumination pathway, and not within the image forming optical elements. This difference should be a major factor to be considered when choosing a microscope configuration for quantitative DIC experiments.

Ergonomic considerations are also important when comparing traditional and de Sénarmont DIC microscope configurations. Modern microscopes are designed with stage and focus control knobs that are positioned on the front of the microscope base, at desktop height, and within easy reach of the operator. These features allow a more relaxed posture with the hands resting comfortably on the desktop to help eliminate fatigue and reduce the possibility of stress, which can ultimately lead to neuromuscular disorders. In a traditional DIC microscope, the operator must continuously raise a hand (and arm) to a level above the objective in order to adjust the position of the Nomarski prism. However, in ergonomic de Sénarmont designs, the compensator assembly is positioned on the microscope base over the front illumination port, very near the lowered focus knobs and stage translational control levers. The close proximity of all microscope control mechanisms on these DIC microscopes dramatically reduces operator fatigue during long periods of continuous observation.

Enhanced video microscopy techniques take advantage of accurate bias retardation values introduced by de Sénarmont compensators. Often referred to by the acronym VE-DIC (video-enhanced differential interference contrast), the technique is capable of recording specimen fine structural detail that is beyond the optical resolution of a compound microscope. Video-enhanced DIC microscopy has been a powerful research tool in cellular biology for measuring the assembly dynamics of individual microtubules and visualizing the motion of bacterial flagella, both of which are not resolved using other techniques. As discussed above, a majority of very thin DIC specimens are best observed at bias retardation values averaging one-twentieth of a wavelength. However, when video-enhanced DIC is employed to image sub-resolution specimens (such as microtubules and miniature organelles), bias retardation values down to approximately one hundredth of a wavelength must be employed. Obviously, these very slight levels of retardation are best achieved with de Sénarmont compensators designed with highly accurate, graduated and vernier-equipped, rotating polarizer (or analyzer) assemblies.

Among the chief imaging advantages of differential interference contrast microscopy, regardless of the mechanism by which bias retardation is introduced, is that (unlike darkfield or phase contrast) the image of smaller specimen features is not obscured by adjoining regions having large optical path gradients. In addition, the shadow-cast appearance of the image on a neutral gray background, coupled to the sensitivity for imaging very small features together with those much larger (for example, minute appendages to living cells or dynamic inclusions and moving organelles within a cell), is a significant improvement over traditional contrast-enhancing techniques. These benefits, in addition to the wide dynamic range of contrast control and shallow depth of field, have all contributed to the widespread popularity of the technique.

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.

Stanley Schwartz - Bioscience Department, Nikon Instruments, Inc., 1300 Walt Whitman Road, Melville, New York 11747.

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.

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

Related Nikon Products

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.

Figure 1 - Transmitted de Sénarmont DIC Microscope Configuration

Several of the major microscope manufacturers are now producing DIC accessory kits for their research-level microscopes that contain the necessary components for using a de Sénarmont compensator instead of a translatable objective prism to introduce bias retardation into the wavefront field. The basic de Sénarmont DIC microscope optical train is presented in Figure 1 for a transmitted light microscope. Semi-coherent bundles of non-polarized white light emitted from localized neighborhoods on the lamp filament (the source is usually a 100-watt tungsten-halogen bulb) first pass through a linear polarizer and quarter-wavelength retardation plate combined together in a de Sénarmont compensator housing, which is attached to the illumination port at the base of the microscope. Linear, elliptical, or circularly polarized light exiting the de Sénarmont compensator next passes through the condenser Nomarski (or Wollaston) beamsplitter prism, where it is first sheared into orthogonal components, and then rendered parallel by the condenser lens system. After passing through the specimen and its surrounding medium, light is gathered by the objective and focused at the interference plane of a second Nomarski prism located above the objective thread seat in the microscope nosepiece. Orthogonal wavefronts are recombined by the objective Nomarski prism, and travel up the optical train to the analyzer, which passes only those components parallel to the transmission azimuth. The parallel wavefronts admitted through the analyzer are able to undergo interference to produce an image that can be observed or recorded by a detector.

In the configuration illustrated in Figure 1, the de Sénarmont compensator is designed to fit and secure tightly onto a mounting bracket positioned inside the large circular knurled wheel that surrounds the field lens and controls the field diaphragm iris size. Mounted above the polarizer in the de Sénarmont compensator housing is a quarter-wavelength retardation plate positioned with the fast optical axis oriented East-West in relation to the microscope frame. In addition, the retardation plate is tilted off-axis by a few degrees from the perpendicular to the optical path in order to reduce reflections, and has a multiple coating of antireflection thin films applied to the upper surface.

The polarizer is contained in a rotatable cylinder sandwiched between the light port housing skirt and the fixed retardation plate (see Figure 2). When the polarizer transmission axis is aligned parallel to the fast axis of the retardation plate, no optical path difference is added to the sheared wavefronts traveling between the condenser and objective prisms (in effect, there is no bias retardation because linearly polarized light exits the compensator). However, the cylinder enclosing the polarizer is designed to be rotated around the microscope optical axis by approximately 180 degrees (plus or minus 90 degrees from the parallel alignment of the polarizer and the retardation plate fast axis) in order to introduce elliptically or circularly polarized light into the optical system. The exact degree of rotation is indicated on some de Sénarmont compensator housings by a graduated scale that contains a zero position (polarizer aligned parallel to the retardation plate) in the center, with linear graduations extending about 45 degrees in both the left and right orientations (as illustrated in Figure 2). Thus, positive and negative bias retardation can be introduced into a de Sénarmont DIC microscope having this type of compensator simply by rotating the polarizer back and forth within its range.

Upon initial installation, the de Sénarmont compensator illustrated in Figure 2 is first aligned with the polarizer transmission azimuth and retardation fast axis oriented in the proper position (East-West) and crossed with respect to the transmission axis of the analyzer (which is oriented North-South). After alignment, the compensator is secured to the light port knob with a locking setscrew. The polarizer axis orientation is marked on the front of the de Sénarmont compensator unit, as described above, and the graduated ruling enables the operator to qualitatively determine the approximate amount of bias retardation introduced into the system when the polarizer is rotated. A locking knob (see Figure 2) can be utilized to hold the polarizer immobile with respect to the quarter-wavelength plate. For brightfield or enhanced contrast techniques without differential interference contrast, the entire polarizer and retardation plate assembly can be removed from the optical path by swinging out the hinged upper section.

Figure 2 - Base Mounted de Sénarmont Compensator

Polarized light having linear, elliptical, or circular characteristics, depending upon the polarizer orientation with respect to the retardation plate, exits the de Sénarmont compensator and next encounters a Nomarski or Wollaston prism positioned within the condenser turret (Figures 1 and 3). DIC condenser prisms, which act as beamsplitters to produce an angular shear to incoming polarized wavefronts, have a similar design characteristics regardless of whether they are intended for use with de Sénarmont compensators or translatable objective prisms. These prisms are usually mounted in a revolving turret designed to house at least three or four individual prism units, similar to the model illustrated in Figure 3. Turret specifications and configurations vary according to the manufacturer, but they generally contain slots for four to eight auxiliary components, including Wollaston or Nomarski prisms, phase contrast annular rings, Hoffman modulation contrast slits, or darkfield light stops. The condenser turret illustrated in Figure 3 contains seven openings, three of which are filled with phase contrast annuli and three with DIC Nomarski prisms. The open slot is employed for brightfield observations.

Each condenser DIC prism (which are also termed compensators or auxiliary prisms) must be specifically matched to a narrow range of objective numerical apertures, so a particular prism may only work for one or two objectives (for example, the 20x and 40x). As a result, between three and five condenser prisms must be utilized to match the entire objective magnification range between 10x and 100x in a typical compound microscope. Some manufacturers design each condenser prism specifically for a particular objective, thus requiring up to seven condenser prisms to span the entire spectrum of dry and oil immersion objectives having varied numerical apertures. The Nikon condenser (Nomarski) prism specifications for de Sénarmont DIC microscopy are catalogued in Table 1, including the prism and objective identification letters, color codes, shear distances, and numerical aperture ranges.

Figure 3 - Universal Condenser Turret DIC Configuration

Condenser DIC prism inserts are constructed with anodized circular aluminum or brass plates having the combination prism (usually oval in shape) secured by optical cement with the shear axis positioned in a fixed, precise orientation. DIC prism wedges are very thin and cut with close tolerances to ensure that angular shear values match those required by the objective numerical aperture. The polished plates must be handled carefully to avoid contamination from fingerprints, oil, dirt, and debris. Each prism frame contains a positioning slot or pin that mates to a corresponding partner in the condenser turret in order to define and secure alignment of the condenser prism with respect to the objective prism and the axes of the polarizer, analyzer, and the de Sénarmont compensator retardation plate. After the prisms are inserted into the condenser turret, they are held firmly in place with a spring or a locking screw. Magnetic or polymer labels with adhesive backings are provided with most universal condensers to allow identification of components in the turret openings after the unit has been assembled.

Universal turret condensers are usually provided with a top lens swing-out mechanism that enables the condenser to be employed with both high (10x through 100x) and low (2x through 5x) magnification objectives. The top lens is placed into the optical path by pulling or pushing the swing lever for the higher magnifications, and removed from the path for use with the lower magnification objectives. A majority of the turret condenser top lens swing arms are threaded to accept lens assemblies designed for both dry and oil immersion objectives. Typically, a dry condenser top lens element will have a numerical aperture value ranging between 0.75 and 0.90, while the corresponding assembly designed for use with oil has a much higher numerical aperture (1.3 to 1.4). Often, there is not enough clearance between the condenser and the microscope stage to allow the swing mechanism to operate properly when the oil top lens element is inserted.

Table 1 - Nikon Condenser Nomarski Prism Specifications

Top Lens

Element

Condenser

Prism Code

Numerical

Aperture Range

Shear Distance

(Micrometers)

Color Code

Dry

DIC L

Less Than 0.5

0.6

Green

Dry

DIC M

0.5 to 1.0

0.3

Green

Dry

DIC H

1.0 or More

0.15

Green

Oil

DIC L

Less Than 0.5

0.6

Yellow

Oil

DIC M

0.5 to 1.0

0.3

Yellow

Oil

DIC H

1.0 or More

0.15

Yellow

Sheared wavefronts exiting the condenser prism are rendered parallel by the condenser optical system and pass through the specimen before being intercepted by the objective front lens element. The optical path difference between orthogonal wavefronts passing through the system (bias retardation) is pre-determined by the position of the polarizer transmission axis with respect to the retardation plate fast axis in the de Sénarmont compensator before light enters the condenser. After experiencing wavefront field distortions by the specimen, light is gathered by the objective optical system and focused onto the interference plane (conjugate to the objective rear focal plane) of a second Nomarski or Wollaston prism positioned above the objective.

In a properly aligned DIC microscope (irrespective of the mechanism utilized to introduce bias retardation), the condenser prism is imaged onto the objective prism by the combined action of the condenser and objective lens systems. As a result, the wavefront shear produced by the condenser prism is exactly matched at every point along the surface of both prisms, which are inverted with respect to one another. Rotating the polarizer in a de Sénarmont compensator produces a wavefront mismatch (bias retardation), which, in turn, introduces an optical path difference that is uniform across the microscope aperture.

Figure 4 - Fixed Nomarski Objective Prisms

In DIC microscopes designed to introduce bias retardation using a de Sénarmont compensator, the objective Nomarski (or in some cases, Wollaston) prisms are secured with precise orientation in fixed mounts that slide into the nosepiece above the objectives, as illustrated in Figures 1 and 4. The Nomarski prisms presented in Figure 4 were imaged between crossed polarizers to illustrate the relative sizes of interference patterns as a function of shear distance (see Table 1). Microscopes using de Sénarmont compensators require a separate objective prism for each objective, but can often utilize the same condenser Nomarski prism for two or more objectives, as described above. The fixed objective prism frames (Figure 4) are easily removed from the optical path for observation in other contrast enhancing modes by sliding the frame out of the slot and away from the microscope nosepiece.

After exiting the objective Nomarski prism, recombined wavefronts next encounter the analyzer (a second polarizer), which is usually placed in a convenient location between the microscope nosepiece and the observation tubes. The analyzer serves to pass wavefront vector components that are parallel to the transmission azimuth and capable of constructive and destructive interference to form a DIC image at the fixed eyepiece diaphragm or camera projection lens. Several microscope nosepiece designs incorporate a slot for insertion of a simple fixed-position linear analyzer oriented North-South with respect to the microscope frame and the polarizer (illustrated in Figure 5(b)). In this configuration, the analyzer is positioned with the transmission axis perpendicular to that of the polarizer (to yield crossed polarizers). Other microscopes utilize fixed analyzers that are inserted into the optical train throughout a number of locations, including intermediate tubes and vertical illuminators (for reflected light configurations). Fixed analyzers are mounted in rectangular frames that can be easily installed or removed from the microscope optical pathway to allow rapid changeover of imaging modes.

Figure 5 - de Sénarmont DIC Microscope Analyzer Configurations

Several analyzer designs feature the same frame style (see Figures 5(a) and 5(b)), but enable rotation of the analyzer element by means of a thumbwheel that is often graduated in 5, 10, 45, or 90-degree increments (Figure 5(a) presents an example). Polarized light microscopes equipped for DIC observation often house the analyzer in an intermediate tube (Figure 5(c) and Figure 6) located between the objective nosepiece and the observation tubes. These units are often designed for precision measurements in polarized light and feature 360-degree graduated vernier scales wrapped around the circumference of the tube (Figure 5(c)), or adjacent to a graduated thumbwheel (Figure 6). In addition, a locking mechanism is included to tightly secure the analyzer in the desired transmission azimuth. In addition, the analyzer is usually mounted on a slider so that it can be conveniently removed from the light path for linearly polarized or brightfield observation. Intermediate tubes for polarized light and DIC microscopy also usually contain a 20 x 6 millimeter DIN standard slot for a quarter-wavelength, full-wave, or de Sénarmont compensator (Figures 5(c) and 6), although several manufacturers use proprietary slot sizes.

An exact determination of the amount of bias retardation introduced to the wavefront field is very difficult to determine in a differential interference contrast microscope that employs a translating Nomarski prism in the objective rear focal plane (traditional Nomarski DIC). For quantitative estimates, Nomarski prism frames having highly accurate micrometer-controlled translation devices have been developed, and these components can be utilized to accurately gauge the amount of bias introduced by moving the prism in small, recorded increments. A more precise determination (approaching one-hundredth of a wavelength) can be made by utilizing a graduated analyzer (or polarizer) coupled to a vernier scale (Figure 5(c) and 6) and a quarter-wavelength retardation plate in a de Sénarmont DIC configuration.

For quantitative determination of bias retardation values, a configuration similar to the one illustrated in Figure 6 is ideal. The intermediate tube presented in Figure 6 is designed to be installed between the objective Nomarski prism in the nosepiece and the microscope observation tubes. Inserted into the lower slot of the tube is a standard 550 nanometer de Sénarmont compensator (originally designed for polarized light measurements) housed in a fixed frame with the fast axis parallel to the polarizer (East-West) and the slow axis parallel to the analyzer (North-South). Above the de Sénarmont compensator is a rectangular frame containing a 360-degree adjustable linear polarizing element (acting as the analyzer), and graduated in single degrees of rotation. The circular control knob is stationed adjacent to a vernier scale that enables accurate determination of the polarizer transmission azimuth orientation, and provides the microscopist with the ability to control the level of bias retardation in the optical system to a few fractions of a wavelength.

Figure 6 - Analyzer Intermediate Tube with Compensator Slot

Intermediate tubes containing a graduated analyzer and a de Sénarmont compensator can be retrofitted onto microscopes originally designed to introduce bias retardation by translation of the objective Nomarski prism (on a sliding frame). After initially configuring the optical system by matching the objective and condenser prisms for maximum overlap of the interference fringes (maximum extinction), the analyzer can be rotated to introduce bias retardation. In fact, microscopes already fitted with the original equipment de Sénarmont compensator (similar or identical to the model presented in Figure 1) can also be configured with the intermediate tube illustrated in Figure 6 (or Figure 5(c)) for quantitative determination of bias retardation. It is important to remove the fixed analyzer if an intermediate tube containing another analyzer is added to the optical system.

In summary, bias retardation in de Sénarmont DIC microscopy can be qualitatively controlled by a simple rotating polarizer unit, as presented in Figures 1 and 2, or with the more quantitative intermediate tubes illustrated in Figures 5(c) and 6. It is not necessary to alter the wavefront field relationship (linear, elliptical, or circularly polarized light) in differential interference contrast prior to entry into the condenser prism. The same effect can be achieved by changing the optical path difference using a quarter-wavelength retardation plate and rotating analyzer after the wavefronts have emerged from the objective prism. In fact, bias retardation can be introduced anywhere in the DIC optical system, provided the change is made somewhere between the polarizer and analyzer, and the proper components are employed with the correct orientation. Traditional DIC microscope designs rely on translation of the objective, or in rare cases, the condenser prism. Newer de Sénarmont compensator microscopes employ a rotating polarizer and a retardation plate near the field lens (primarily for ergonomic measures) to achieve the same effect.

Modern polarizing and DIC microscopes position the polarizer and analyzer in strategic locations with respect to the field lens, condenser, objective, and observation tubes. In older microscopes, these polarizing elements can be found installed in a wide variety of locations. It should be noted, however, that placing polarizing elements in or very near a conjugate image plane (the field diaphragm, specimen stage, or eyepiece fixed aperture) is not a good idea, because scratches, imperfections, dirt, and debris on the glass or polymer surface can be imaged along with the specimen.

de Sénarmont DIC for Inverted Microscopes

Inverted transmitted light (tissue culture) microscopes are often equipped with DIC optical components to aid in the visualization, photography, and digital imaging of a variety of transparent specimens, including living cells, embryos, and tissue slices. Both traditional and de Sénarmont DIC optical systems have been adapted for use with inverted microscopes. The major difference between DIC configurations in upright and inverted microscopes is the condenser system, which generally requires specialized long working distance optical components for the inverted instruments. In many cases, inverted microscope objectives must also be designed to operate with long or extra-long working distances to match condenser aperture illumination cones.

Figure 7 - de Sénarmont DIC for Inverted Microscopes

The most popular inverted microscope configuration places the objective revolving nosepiece beneath the mechanical stage, where it is attached to the main body through an intermediate tube or direct port to the main internal optical train. In some models, fixed Nomarski DIC prisms on slider frames (see Figure 4), which are identical to the prisms used in upright microscopes, can be inserted into the nosepiece. Other microscopes employ a single sliding Nomarski prism, mounted in a long rectangular frame, which is used for all objectives (10x through 100x; traditional Nomarski DIC). The Nomarski prism frame is mounted beneath the nosepiece in an intermediate tube that enables the beamsplitter to be easily removed from the optical pathway for brightfield, phase contrast, Hoffman modulation, or other imaging modes. Bias retardation is introduced by means of a control knob positioned at the end of the prism frame. In the latter configuration, a single Nomarski prism serves to recombine wavefronts for all of the objectives in the nosepiece, although many microscope designs require axial relocation of the prism at higher magnifications to accommodate variations in the location of the objective rear focal plane.

A typical inverted microscope designed for differential interference contrast using a de Sénarmont compensator is presented in Figure 7. Semi-coherent light waves emitted by the tungsten-halogen lamp on the illumination pillar first pass through a cascade of filters (neutral density, color balancing, and interference) before being deflected by a right-angle prism through the field lens and into the condenser system. Attached on top of the condenser is a de Sénarmont compensator (see Figure 8) consisting of a linear polarizer and a quarter-wavelength retardation plate. After light passes through the de Sénarmont compensator, it is focused at the condenser aperture iris diaphragm (site of the condenser focal plane), which is conjugate with the interference plane of a Nomarski prism located in the condenser turret (Figure 8). Sheared wavefronts are focused parallel to each other by the condenser lens system and illuminate the specimen before being collected by the objective front lens, which is positioned beneath the specimen stage.

A second Nomarski prism (illustrated in Figure 7) is housed beneath the objective in the microscope nosepiece, and serves to recombine sheared wavefronts after they are deformed by the specimen and focused by the objective. The recombined waves then pass through a second polarizer (the analyzer; see Figure 7) located in a sliding rectangular frame and inserted into an intermediate tube or slot between the nosepiece and the microscope base. After leaving the analyzer, the wavefronts (which are now capable of undergoing destructive and constructive interference) traverse through the microscope internal optical train to form an image at the fixed diaphragm of the eyepieces or the projection lens of a traditional or digital camera system.

Figure 8 - Inverted Microscope de Sénarmont DIC Condenser

As discussed above, microscopes equipped for DIC with de Sénarmont compensators have fixed objective Nomarski prisms mounted into frames similar to those illustrated in Figure 4. Bias retardation is introduced into the optical system by rotating the polarizer transmission azimuth on the de Sénarmont compensator (Figure 8) attached to the condenser. The Nomarski condenser prisms are mounted in a turret, similar to the design for upright microscopes (see Figure 3), but with a different frame design. As is the case for upright microscopes, the condenser Nomarski prisms are specifically matched to a narrow range of objective numerical apertures, so several prisms must be employed to cover the entire magnification range (Table 1).

Condenser prisms (usually circular in shape) are mounted with a precise orientation of the shear axis, using optical cement, into pie-shaped aluminum wedges that bolt into the condenser turret. Analogous to upright microscope condenser prisms, the inverted microscope Nomarski prisms are very thin and cut with close tolerances to ensure that angular shear values match those required by the objective numerical aperture. The condenser turret can accept between three and five wedges, which is enough to cover the entire microscope magnification range. In addition to Nomarski prisms, the condenser turret can accommodate phase contrast annulus plates and Hoffman modulation contrast slit plates. Any combination of phase annuli, slit plates, and Nomarski prisms can be utilized in the condenser.

First-Order Compensation Plates

Differential interference contrast microscopy with de Sénarmont compensators enables the introduction of bias retardation values between plus and minus one-half wavelength. In order to increase the path difference between sheared wavefronts, a full-wave retardation plate can be added to the optical system (yielding retardation values up to 1.5 times the wavelength of green light). Compensating plates bestow greater control for adjusting the contrast of specimen details in relation to the background intensity and color values, and also enable more precise tuning of the bias retardation between wavefronts. In addition, full-wavelength compensators are frequently employed for optical staining of thicker transparent specimens, which are normally imaged over a limited range of grayscale intensities.

On upright or inverted microscopes equipped with a de Sénarmont compensator for DIC observations, a full-wave retardation plate can be inserted into the optical pathway between either the objective prism and the analyzer or the de Sénarmont compensator and the condenser prism. Upright microscopes usually have a slot beneath the condenser or in an intermediate tube above the objectives, which is designed to receive a full-wave retardation plate. Inverted microscopes are more restricted (due to design constraints) in the placement of auxiliary compensators. The inverted microscope condenser illustrated in Figure 8 can accept a full-wavelength retardation plate in a slot beneath the de Sénarmont compensator housing, but does not have a provision for introducing auxiliary compensators between the objective and analyzer. A majority of the inverted microscope condensers offered by other manufacturers are similar in design.

In conclusion, de Sénarmont compensators enable more control of bias retardation in DIC microscopy than do the traditional Nomarski objective prism designs that rely on translation of the prism across the microscope optical axis. By coupling a graduated polarizer (or analyzer) to a fixed quarter-wavelength retardation plate, the amount of bias retardation introduced into the optical system can be determined with a high degree of precision. Finally, microscope designs that incorporate a de Sénarmont compensator near the field lens at the base of the instrument have ergonomic advantages over traditional configurations.

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.

Stanley Schwartz - Bioscience Department, Nikon Instruments, Inc., 1300 Walt Whitman Road, Melville, New York 11747.

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.

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

Related Nikon Products

Microscope Optical Path

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.

A schematic cutaway diagram of the key optical train components in a reflected light differential interference contrast microscope is presented in Figure 1. Illumination generated by the light source passes through the aperture and field diaphragms (not illustrated) in a vertical (episcopic) illuminator before encountering a linear polarizer positioned with the transmission axis oriented East-West with respect to the microscope frame. Linearly polarized light exiting the polarizer is reflected from the surface of a half-mirror placed at a 45-degree angle to the incident beam. The deflected light waves, which are now traveling along the microscope optical axis, enter a Nomarski prism housed above the objective in the microscope nosepiece where they are separated into polarized orthogonal components and sheared according to the geometry of the birefringent prism.

Acting in the capacity of a high numerical aperture, perfectly aligned, and optically corrected illumination condenser, the microscope objective focuses sheared orthogonal wavefronts produced by the Nomarski prism onto the surface of an opaque specimen. Reflected wavefronts, which experience varying optical path differences as a function of specimen surface topography, are gathered by the objective and focused on the interference plane of the Nomarski prism where they are recombined to eliminate shear. After exiting the Nomarski prism, the wavefronts pass through the half-mirror on a straight trajectory, and then encounter the analyzer (a second polarizer) positioned with the transmission axis oriented in a North-South direction. Components of the orthogonal wavefronts that are parallel to the analyzer transmission vector are able to pass through in a common azimuth, and subsequently undergo interference in the plane of the eyepiece fixed diaphragm to generate amplitude fluctuations and form the DIC image. Formation of the final image in differential interference contrast microscopy is the result of interference between two distinct wavefronts that reach the image plane slightly out of phase with each other, and is not a simple algebraic summation of intensities reflected toward the image plane, as is the case with other imaging modes.

A significant difference between differential interference contrast in transmitted and reflected light microscopy is that two Nomarski (or Wollaston) prisms are required for beam shearing and recombination in the former technique, whereas only a single prism is necessary in the reflected light configuration. Light passes through the same Nomarski prism twice, traveling in opposite directions, with reflected light DIC. The shear produced when the light waves pass through the prism on the way to the objective is cancelled during their second journey through the prism upon returning from the specimen surface. In this regard, the Nomarski prism and objective serve an identical function for incoming light waves as the first prism and condenser optical system in a transmitted light microscope. Similarly, light reflected from the specimen surface is gathered by the objective and focused into the Nomarski prism interference plane (conjugate to the objective rear focal plane), analogous to the manner in which these components function in transmitted light.

The optical pathway, both for the entire wavefront field and a single off-axis light ray, in reflected light DIC microscopy are illustrated in Figures 2(a) and 2(b), respectively. In each case, linearly polarized light from the polarizer is deflected by the half-mirror and enters the Nomarski prism located behind the objective. Sheared wavefronts are focused by the objective lens system and bathe the specimen with illumination that is reflected in the form of a distorted wavefront (Figure 2(a)) or the profile of an opaque gradient (Figure 2(b)) back into the objective front lens. The optical path difference produced between orthogonal wavefronts enables some of the recombined light to pass through the analyzer to form a DIC image. In Figure 2(b), note that the trajectory of the light ray incident on the specimen is displaced by the same distance from the microscope optical axis as the ray reflected from the surface.

The Wollaston and Nomarski prisms employed in reflected light DIC microscopy are fabricated in the same manner as those intended for use with transmitted light instruments. The single birefringent prism for reflected light is comprised of two precisely ground and polished wedge-shaped slabs of optical quartz that are identical in shape, but have differing orientations of the optical axes. In a Wollaston prism, the quartz wedges are cemented together at the hypotenuse with an orientation that positions the optical axes perpendicular to each other. Conversely, in a Nomarski prism, the axis of one wedge is parallel to the flat surface, while the axis of the other wedge is oriented obliquely. As a result of geometrical constraints, the interference plane for a Wollaston prism lies near the center of the junction between the quartz wedges (inside the compound prism), but the Nomarski prism interference plane is positioned at a remote location in space, outside the prism itself. Incident linearly-polarized light waves (parallel to the optical axis of the microscope) that enter a Wollaston or Nomarski prism are divided into two mutually perpendicular (orthogonal) components, termed the ordinary and extraordinary wave, which have identical amplitudes (70.7 percent of the original polarized wave) and are coherent (provided, of course, that the illumination source is also coherent). In order to produce orthogonal components having equal amplitudes, the linearly polarized light entering a Nomarski or Wollaston prism is oriented with the electric vector vibration direction positioned at a 45-degree angle with respect to the principal optical axis in the upper wedge of the prism.

Figure 2 - Reflected DIC Microscope Optical Pathways

An angular splitting or shear of the orthogonal wavefronts occurs at the boundary between cemented quartz wedges in a Wollaston prism, and the waves become spatially separated by an angle defined as the shear angle. At this boundary, the ordinary and extraordinary waves also exchange identities and diverge away from each other as a function of the refractive index experienced by each wave as it travels through the quartz prism. The shear angle and separation distance is constant for all incident wavefronts across the face of the prism, regardless of the entry point. The direction of wavefront shear is defined by the prism shear axis, which lies in the plane of the Wollaston prism and is parallel to the optical axis of the lower quartz wedge section. In a Nomarski prism, the wedge having an oblique optical axis produces wavefront shear at the quartz-air interface, and is responsible for defining the shear axis.

Nomarski and Wollaston prisms not only separate linearly polarized light into two orthogonal components, they also produce a relative phase shift (often termed an optical path difference) in each wavefront relative to the other. The degree of phase shift between the wavefronts varies linearly with the location of the input light beam in relation to the shear direction. Thus, the prism can be laterally translated along the optical axis of the microscope in the shear direction (a process known as introduction of bias retardation) to enable adjustment of the optical path difference introduced between the orthogonal wave components. In this manner, fine-tuning of the relative intensity in the image can be manipulated to produce the distinctive shadow-cast appearance for which DIC microscopy is so well known. Images appear as if they were illuminated from a highly oblique light source originating from a single azimuth.

Because the interference plane in a conventional Wollaston prism is positioned in the central portion of the prism, at approximately the centerline between the two quartz wedges, it is difficult to adapt this prism design for use with standard microscope objectives in reflected light DIC microscopy. This problem arises because the interference plane of the prism must coincide and overlap with the rear focal plane of the objective, which often lies below the thread mount inside a glass lens element. On the other hand, external displacement of the interference plane in Nomarski prisms renders them ideal for use with microscope objectives since they can be positioned some distance away (for example, in the nosepiece) and still establish a conjugate relationship between the objective rear focal plane and the compound prism interference plane. In a reflected light DIC microscope, the Nomarski prism is oriented so that the interference plane is perpendicular to the optical axis of the microscope (as is the objective rear focal plane).

The ordinary and extraordinary wavefronts proceeding to the specimen through a Nomarski prism experience optical path differences that have a magnitude dependent upon the location of the wave as it enters the prism. After the wavefronts exit the prism, they enter the objective lens system (acting as an illumination condenser) from the rear, and are focused into a parallel trajectory before being projected onto the specimen. Reflection of the orthogonal wavefronts from a horizontal, opaque specimen returns them to the objective, but on the opposite side of the front lens and at an equal distance from the optical axis (see Figure 2(b)). The waves gathered by the objective are focused on the Nomarski prism interference plane (again on the opposite side from their journey down), which results in a phase shift that exactly offsets the original difference produced before the waves entered the objective. As a result, the positional exchange of incident and reflected waves results in cancellation of relative phase shifts across the entire microscope aperture. A system of this type is referred to as being self-compensating, and the image produced has a uniform intensity. Compensation of the reflected light DIC system can be compared to that for transmitted light, where two matched, but inverted, Nomarski (or Wollaston) prisms are used to shear and recombine the beam. In the transmitted light configuration, the condenser prism (often termed the compensating prism) is imaged onto the objective prism (referred to as the principal prism) so that optical path differences are matched at every point along the surface of the prisms. Thus, in the transmitted light configuration, the principal and compensating prisms are separate, while the principal prism in reflected light DIC microscopy also serves the function of the compensating prism.

Light waves employed for reflected DIC microscopy must be at least moderately collimated in order to provide uniform compensation across the full beamwidth for the two required passes through the prism, and to insure that phase differences introduced by slopes and reflection boundaries in the specimen can be detected. Because the phase difference experienced by a beam on its first pass through the prism is governed by the pathway, accurate compensation of the reflected beam requires passage along a complimentary portion of the prism. In order to ensure collimation of the light beam, the microscope must be properly configured for Köhler illumination to guarantee that input waves are parallel (or nearly so) to the optical axis. A poorly collimated input beam will result in nonuniform compensation across the prism (and the resulting image), and destroys the unique phase relationship between orthogonal components at each image point.

Anatomy of the Reflected Light Microscope

The vertical illuminator is a key component in all forms of reflected light microscopy, including brightfield, darkfield, polarized light, fluorescence, and differential interference contrast. A reflected light (often termed coaxial, or on-axis) illuminator can be added to a majority of the universal research-level microscope stands offered by the manufacturers. Reflected light DIC can be performed using the Nikon LV100N POL upright microscope. The primary function of a vertical illuminator is to produce and direct semi-coherent and collimated light waves to the rear aperture of the microscope objective and, subsequently, onto the surface of a specimen. Reflected light waves gathered by the objective then travel a pathway similar to the one utilized in most transmitted light microscopes. A critical component of the vertical illuminator is a partially reflecting plane glass mirror (referred to as a half-mirror; see Figure 3) that deflects light traveling from the horizontal illuminator by 90 degrees into the vertical optical train of imaging components in the microscope. The half-mirror, which is oriented at a 45-degree angle with respect to both the illuminator and microscope optical axis, also allows light traveling upward from the objective to pass through undeviated to the eyepieces and camera system.

Modern vertical illuminators designed for multiple imaging applications usually include a condensing lens system to collimate and control light from the source. In addition, these illuminators contain an aperture iris diaphragm and a pre-focused, centerable field diaphragm to enable the microscope to operate in Köhler illumination (Figure 3). Vertical illuminators also have numerous slots and openings for insertion of light balancing and neutral density filters, polarizers, compensators, and fluorescence filter combinations housed in cube-shaped frames. When configured to operate with infinity-corrected objectives, vertical illuminators are equipped with a tube lens (see Figure 1) to focus light waves into the intermediate image plane. Both tungsten-halogen and arc-discharge lamphouses can be utilized with vertical illuminators (often interchangeably) to provide a wide range of illumination intensity and spectral characteristics.

Figure 3 - Reflected Light Microscope Vertical Illuminator Anatomy

The most popular choice of a light source for reflected light microscopy (including the DIC imaging mode) is the ubiquitous tungsten-halogen lamp, which features a relatively low cost and long lifespan. Incandescent halogen lamps are moderately bright, but require color balancing filters to raise their color temperature to daylight levels for digital imaging and traditional photomicrography with film. An alternative choice, useful at high magnifications and very low bias retardation values (where illumination intensity is critical), is the 75 or 150-watt xenon arc-discharge lamp. Xenon lamps feature a high level of brightness across the entire visible light spectrum, and have color a temperature output that approximates the value required for daylight balance.

When white light from a tungsten-halogen or arc-discharge lamp is used for illumination in reflected light DIC microscopy, the interference fringes associated with topographical changes in the specimen can actually appear in narrow rainbow patterns along the features as the various colors destructively interfere at slightly different locations on the surface. Use of a narrower wavelength band of illumination in specialized applications (for example, light emitted from a laser) will produce a DIC image where the fringes are established by the interference of a single wavelength. These fringes will be sharper and more defined, and their location will not depend upon the spectral response of the detector.

Illustrated in Figure 4 are images of the region near a bonding wire pad on the surface of a microprocessor integrated circuit captured in brightfield, darkfield, and differential interference contrast illumination using a vertical illuminator and reflected light. The brightfield image (Figure 4(a)) suffers from a significant lack of contrast in the circuit details, but provides a general outline of the overall features present on the surface. Darkfield illumination (Figure 4(b)) reveals only slightly more detail than brightfield, but does expose discontinuities near the vertical bus lines (central right-hand side of the image) and the bonding pad edges on the left. The differential interference contrast image (Figure 4(c)) yields a more complete analysis of the surface structure, including the particulate bonding pad texture, connections from the bonding pad to the bus lines, and numerous fine details in the circuitry on the left-hand side of the image.

Figure 4 - Integrated Circuit in Brightfield, Darkfield, and DIC with Reflected Light

Köhler illumination in reflected light microscopy relies on two variable diaphragms positioned within the vertical illuminator. The basic system is configured so that an image of the lamp filament is brought into focus at the plane of the aperture diaphragm, which is conjugate to the rear focal plane of the objective (where the filament can also be observed simultaneously in focus). A field diaphragm, employed to determine the width of the illumination beam, is positioned in the same conjugate plane as the specimen and the fixed diaphragm of the eyepiece. The aperture iris diaphragm is closer to the light source, while the field diaphragm is closer to the objective (the opposite configuration from that employed for transmitted illumination). Differential interference contrast is particularly dependent upon Köhler illumination to ensure that the waves traversing the Nomarski prism are collimated and evenly dispersed across the microscope aperture to produce a high level of contrast.

On most reflected light microscopes, the field diaphragm can be centered in the optical pathway by partially closing the iris aperture and translating the entire diaphragm via a set of centering screws (or knobs) adjacent to the aperture opening control lever. In practice, the field diaphragm should be opened until it is just outside the viewfield or the area to be captured on film or in a digital image. The primary purpose of the field diaphragm is to control the size of the field of view and to prevent stray light from obscuring specimen details. In conjunction with the field diaphragm, the aperture diaphragm determines the illumination cone geometry and, therefore, the angle of light striking the specimen from all azimuths. The iris diaphragm size can be modulated to adjust specimen contrast, and generally should be set to a size that is between 60 and 80 percent of the objective rear aperture. Such a setting provides the best compromise between maximum resolution and acceptable contrast.

Reflected Light DIC Microscope Configuration

Because the components for differential interference contrast must be precisely matched to the optical system, retrofitting an existing reflected light microscope, which was not originally designed for DIC, is an undesirable approach. Instead, all of the major microscope manufacturers now offer industrial and research-grade microscopes equipped with vertical illuminators and the necessary auxiliary optical components (usually marketed in kits) to outfit a microscope for DIC observation. Several different approaches to instrument design have yielded two alternatives for the introduction of bias retardation into the differential interference contrast microscope optical system.

The traditional method for establishing reflected light DIC is to employ a Nomarski prism attached to a mobile carriage within a rectangular frame (often termed a slider) that fits into the microscope nosepiece base, above the revolving objective turret (Figures 5(a) and 5(b)). In this design, bias retardation is introduced by rotating a thumbwheel positioned at the end of the slider that, in turn, translates the Nomarski prism back and forth laterally across the microscope optical axis. An alternative technique, termed de Sénarmont compensation (see Figure 6), utilizes individual fixed prisms for each objective (Figure 5(d)), and a quarter-wavelength retardation plate in combination with the linear polarizer (Figure 5(c)) to introduce an optical path difference (bias retardation) between orthogonal wavefronts.

Figure 5 - Reflected Light DIC Microscope Optical Components

All microscope designs that employ a vertical illuminator for reflected light observation suffer from the problem of stray light generated by the reflections from the illuminator at the surface of optical elements in the system. In particular, the upper and lower planar surfaces of the Nomarski prism can be problematic in producing annoying reflections that create excessive glare and degrade image quality. To counter this effect, Nomarski prisms designed for reflected light microscopy are fabricated so that the interference plane is positioned at an angle with respect to the shear axis of the prism (see Figure 2(b)). When the interference plane of the specialized Nomarski prism is brought into coincidence with the objective rear focal plane (perpendicular to the microscope optical axis) by its positioning inside the sliding frame or fixed housing, the flat outer wedge surfaces are now inclined with respect axial illumination pathway (Figures 1, 2(b), and 5(a)). As a result, reflections are diverted away from the half-mirror, specimen, eyepieces, and camera system so as not to adversely affect image intensity and contrast.

In modern microscopes, the distance between the objective focal plane and the seating face on the nosepiece is a constant value, often referred to as the parfocal distance. Therefore, a single Nomarski prism can often be mounted at a fixed distance from the objective seats (and rear focal planes) on the nosepiece in a slider frame, and service the entire magnification range with regards to beam shearing and recombination duties. In some cases, especially at the higher magnifications, variations in the position of the objective rear focal plane can be accommodated by axial translation of the Nomarski prism within the slider (illustrated in Figures 5(a) and 5(b)). This is often accomplished with a knob or lever that relocates the entire prism assembly up and down along the microscope optical axis. Reflected light microscopes that utilize a single prism for DIC are able to introduce bias retardation by laterally translating the prism across the microscope optical axis with a thumbwheel. The entire Nomarski prism slider can be removed from the optical path when the microscope is used for other imaging modes (brightfield, polarized light, darkfield, and fluorescence).

Microscopes equipped with a single translatable Nomarski prism in the nosepiece require only a polarizer and an analyzer as accompanying components in order to operate in differential interference contrast imaging mode. The polarizer is usually mounted together with a rack-and-pinion or planetary gearset into a thin rectangular frame, so that the transmission azimuth can be rotated through 360 degrees with a thumbwheel. The polarizer frame is introduced into the light path between the field diaphragm and the half-mirror through a slot in the vertical illuminator. Likewise, the analyzer can also be housed in a frame that enables rotation of the transmission axis. Analyzer frames are usually placed into a slot in the nosepiece or near the tube lens in the upper portion of the vertical illuminator. In some cases, either the analyzer or polarizer is mounted in a fixed frame that does not allow rotation, but most microscopes provide the operator with the ability to rotate the transmission azimuth of at least one of the polarizers in order to compensate for opaque specimens that absorb light. Housing the polarizer and analyzer in slider frames enables the operator to conveniently remove them from the light path for other imaging modes. When the polarizers remain in place and the Nomarski prism slider is removed, the microscope is configured for observation in polarized reflected light mode.

An alternative mechanism for introduction of bias retardation into the reflected light DIC microscope optical system is to couple a de Sénarmont compensator in the vertical illuminator with fixed-position Nomarski prisms (illustrated in Figures 5(c), 5(d), and 6) for the objectives. In the de Sénarmont configuration, each objective is equipped with an individual Nomarski prism designed specifically with a shear distance to match the numerical aperture of that objective. The prisms are glued into frames and housed in a dust-tight assembly that mounts between the objective and the microscope nosepiece (Figure 5(d)). Objectives are threaded into the Nomarski prism housing, which is then secured to the nosepiece. A small lever is used to shift the prism frame into and out of the optical pathway (the prism positionlever in Figure 5(d)). Because of the increased number of Nomarski prisms required for the de Sénarmont DIC microscope configuration, these accessories are considerably more expensive than the sliding prism in a traditional reflected light Nomarski DIC microscope.

The optical train of a reflected light DIC microscope equipped with de Sénarmont compensation is presented in Figure 6. Light from the illumination source is focused by the collector lens and passes through the aperture and field diaphragms before encountering a linear polarizer in the vertical illuminator. Positioned directly behind the polarizer in the optical pathway is a quarter-wavelength retardation plate fixed into position where the fast axis is oriented East-West with respect to the microscope frame. Together, the polarizer and retardation plate comprise the de Sénarmont compensator (Figure 5(c)). When the polarizer transmission azimuth is aligned parallel to the fast axis of the retardation plate in the de Sénarmont compensator, linearly polarized light emerges from the assembly, and is deflected at a 90-degree angle by the vertical illuminator half-mirror into the pathway of imaging elements in the microscope.

Figure 6 - de Sénarmont Reflected Light DIC Configuration

After the polarized light waves reach the half-mirror and are deflected, the remainder of the microscope optical train operates in a manner similar to that of a traditional DIC reflected light microscope. Thus, on the downward journey through the reflected light microscope, linearly polarized light first encounters the fixed Nomarski prism and is sheared according to the geometry of the prism wedges. After being focused by the objective lens elements and projected onto the opaque specimen, light is reflected back into the objective where it converges at the rear focal plane (coincident with the Nomarski prism interference plane). Sheared wavefronts are recombined at the prism interference plane and proceed to the analyzer, where components that are parallel to the transmission azimuth are passed on to the intermediate image plane. At the image plane, constructive and destructive interference occurs between wavefronts emerging from the analyzer to generate the DIC image.

Bias retardation is introduced into the reflected light de Sénarmont DIC system simply by rotating the linear polarizer in the vertical illuminator. By rotating the polarizer transmission azimuth with respect to the fast axis of the retardation plate, elliptically and circularly polarized light having an optical path difference between the orthogonal wavefronts is produced. When the polarizer axis is rotated up to 45 degrees in one direction, right-handed elliptical or circular polarizer light emerges from the de Sénarmont compensator. Rotating the polarizer in the opposite direction produces elliptical or circular wavefronts having a left-handed rotational sense. The optical path difference introduced by rotating the polarizer (over a range of plus or minus one-half wavelength) is further compounded when the orthogonal wavefronts enter the Nomarski prism and are sheared across the face of the prism. Introducing an optical path difference at the de Sénarmont compensator is analogous to the effect achieved when the objective Nomarski prism is translated across the optical path in a traditional DIC microscope configuration.

Characteristics of Reflected Light DIC Specimens and Images

In reflected light DIC microscopy, the optical path difference produced by an opaque specimen is dependent upon the topographical geometrical profile (surface relief) of the specimen and the phase retardation that results from reflection of sheared and deformed orthogonal wavefronts by the surface. For a majority of the specimens imaged with DIC, the surface relief varies only within a relatively narrow range of limits (usually measured in nanometers or micrometers), so these specimens can be considered to be essentially flat with shallow optical path gradients that vary in magnitude across the extended surface. Phase changes occurring at reflection boundaries present in the specimen also produce and optical path difference that leads to increased contrast in the DIC image. These phase differentials are more likely to be found at junctions between different media, such as grain boundaries and phase transitions in metals and alloys, or aluminum and metal oxide regions in a semiconductor integrated circuit.

Although reflected light DIC microscopy has been heavily employed for examination of metallographic specimens for the past few years, currently the most widespread and significant application is the examination of semiconductor products as a quality control measure during the fabrication process. In fact, most of the manufacturers now offer microscopes designed exclusively for examination of integrated circuit wafers in DIC, brightfield, and darkfield illumination. DIC imaging enables technicians to accurately examine large volumes of wafers for defects that are not revealed by other microscopy techniques (as illustrated in Figure 4). Minute variations in the geometrical profile of the wafer surface appear in shadowed relief, and maximum image contrast is achieved when the Nomarski prism setting is adjusted to render the background a neutral gray color.

When the Nomarski prism is translated along the microscope optical axis in a traditional reflected light DIC configuration, or the polarizer is rotated in a de Sénarmont instrument, an optical path difference is introduced to the sheared wavefronts, which is added to the path difference created when the orthogonal wavefronts reflect from the surface of the specimen. The net result is to render the specimen image in pseudo three-dimensional relief where regions of increasing optical path difference (surface relief or reflection boundaries) appear much brighter or darker, and those exhibiting decreasing path length appear in reverse. Distinguishing features on the specimen surface appear similar to elevated plateaus or sunken depressions, depending on the gradient orientation or reflection characteristics. Unlike the situation with transmitted light DIC, the three-dimensional appearance often can be utilized as an indicator of actual specimen geometry where real topographical features are also sites of changing phase gradients.

The shadow-cast orientation is present in almost every image produced by reflected light DIC microscopy after bias retardation has been introduced into the optical system. In addition, the direction of optical shear is obvious and can be defined as the axis connecting regions of the image displaying the highest and lowest intensity values. Surface features become distinguishable because shadow directions are often reversed for specimen details that posses either a higher or lower topographical profile than the surrounding surface. Because the shear axis is fixed by Nomarski prism design and other constrains involved in wavefront orientation for reflected light DIC microscopy, the axis direction cannot be altered to affect specimen contrast through a simple setting on the microscope. However, the relative phase retardation between sheared wavefronts can be reversed by relocating the Nomarski prism from one side of the microscope optical axis to the other (shifting the bias retardation value from negative to positive, or vice versa). The same maneuver can be accomplished by rotating the polarizer to the corresponding negative value on a de Sénarmont compensator. When phase retardation is altered as just described, the orientation of bright and dark edges in the image is reversed by 180 degrees.

Orientation Effects on Reflected Light DIC Images

As discussed above, reflected light DIC images are inherently bestowed with a pronounced azimuthal effect, which is the result of asymmetrical orientation of the beamsplitting Nomarski prism with respect to the microscope optical axis and the polarizers. The result is that many opaque specimens imaged in differential interference contrast have a prerequisite orientation limitation in order to achieve maximum contrast (either parallel or perpendicular to the shear axis) that restricts freedom of specimen rotation. This is especially critical with highly ordered semiconductors having numerous extended, linear regions intermixed with closely-spaced periodic structures.

Figure 7 - Azimuth Orientation Effects in Reflected Light DIC Microscopy

Azimuth contrast effects in reflected light differential interference contrast can be utilized to advantage by equipping the microscope with a 360-degree rotating circular stage. An essential element in polarized light microscopy, circular stages enable the operator to rotate the specimen with respect to the shear axis in order to maximize or minimize contrast effects for selected specimen features. Contrast in reflected light DIC microscopy achieves a minimum level for linear phase specimens that extend along the direction of shear, but can be varied significantly by rotating the stage by 90 degrees. Non-linear metallurgical specimens, such as mosaic grain boundaries, wires, amorphous alloys, and crystalline spherulites, do not display significant azimuthal effects in reflected light DIC, and can usually be imaged satisfactorily in a variety of orientations.

Presented in Figure 7 are two semiconductor integrated circuit specimens, each having a significant amount of periodicity, but displaying a high degree of asymmetry when imaged in reflected light DIC. Figures 7(a) and 7(b) illustrate the same region of a microprocessor arithmetic logic unit located near the pad ring, which contains numerous bus lines, bonding wire pads and registers. When the circuit is positioned with the long axis of the bonding pad oriented perpendicular to the shear axis (northwest to southeast for all images in Figure 7), the central trapezoid-shaped region of bus lines becomes very dark and almost extinct (Figure 7(a)), losing virtually all recognizable detail. Rotating the integrated circuit by 90 degrees (Figure 7(b)), highlights the central trapezoid bus structure, but causes adjacent areas to lose contrast. In order to capture all the detail present on the surface of this integrated circuit, the optimum orientation is to position the elongated bus structure at a 45-degree angle to the shear axis of the microscope.

Several mask alignment markers are illustrated in the image of a semiconductor surface presented in Figure 7(c). One of the markers has been placed on a metallic bonding pad, while the other rests on a smooth metal oxide surface. Both markers contain eight lines, equally spaced at 45-degree intervals, and having the same length. Note that the lines oriented parallel to the shear axis are very dark for the marker resting on the metal oxide surface, while the upper left line on the bonding pad marker is almost invisible. The marker lines oriented perpendicular (northeast to southwest) to the shear axis are much brighter and far more visible than lines having other orientations, although the lines parallel and perpendicular to the image boundaries are clearly visible.

The correlation between image contrast and specimen orientation in reflected light DIC microscopy can often be utilized to advantage in the investigation of extended linear structures (especially in semiconductor inspection). By capturing images at several orientations, DIC microscopy is often able to present a clear representation of the complex morphology present in many extended, linear specimens. In addition, when optical sectioning methodology is coupled to azimuth-specific imaging, reflected light DIC microscopy can often reveal features that are difficult, or impossible, to distinguish using alternative techniques.

Optical Staining in Reflected Light DIC

Bias retardation between the sheared wavefronts in reflected light DIC microscopy can be manipulated through the use of compensating plates, such as a first-order (often termed a full-wave or first-order red) plate having a retardation value equal to a full wavelength in the green region (550 nanometers) of the visible light spectrum. Compensating plates bestow greater control for adjusting the contrast of specimen details in relation to the background intensity and color values, and also enable more precise tuning of the bias value between orthogonal wavefronts. These birefringent components are also frequently employed for optical staining of opaque specimens, which are normally rendered over a limited range of grayscale values.

Figure 8 - Optical Staining in Reflected Light DIC Microscopy

Optical staining is accomplished either through translation of the Nomarski prism across the optical pathway by a significant distance from maximum extinction, or by inserting a full-wave compensator behind the quarter-wavelength retardation plate in a de Sénarmont configuration. A full range of interference colors can be observed in specimen details when the Nomarski prism is translated to extreme ranges, or the polarizer is rotated with de Sénarmont compensation coupled to a full-wave plate. With the compensator in place, the background appears magenta in color, while image contrast is displayed in the first-order yellow and second-order blue colors of the Newtonian interference color spectrum. Under these conditions, small variations in bias retardation obtained by translation of the Nomarski prism (or rotating the polarizer in a de Sénarmont compensator) yield rapid changes to interference colors observed in structures having both large and small surface relief and reflection phase gradients.

Illustrated in Figure 8 are three specimens imaged in reflected light DIC with a full-wave retardation plate inserted behind the de Sénarmont compensator in a fixed-prism microscope configuration. Figure 8(a) reveals surface defects in a high-temperature superconducting ceramic single crystal grown from an amorphous flux at 950 degrees Celsius. Although twinning defects in the crystal are difficult to discern without applying optical staining techniques, these crystalline mishaps become quite evident and are manifested by significant interference color fluctuations when the retardation plate is installed. Similarly, adhesion failure in a magnetic thin film is clearly imaged when optical staining techniques are employed in reflected light DIC (Figure 8(b)). Separation points in the film are imaged as wrinkles that appear in spectacular relief, surrounded by interference fringes, when observed in white light. Finally, bus line details stand out in sharp color contrast on the surface of the integrated circuit presented in Figure 8(c). A wide spectrum of differential color effects are possible with integrated circuits in reflected light DIC microscopy, based on a number of factors, including the presence or absence of silicon nitride or polyimide protective coatings, phase relationships between fabrication materials, and the feature linewidth of the fabrication process.

Although optical staining is also possible in transmitted light DIC, the effect is far more useful with reflected light techniques, especially when examining flat, planar specimens, such as integrated circuits that have surface relief variations restricted to relatively narrow limits. In contrast to the transparent specimens imaged with transmitted light, surface relief in opaque specimens is equivalent to geometrical thickness. In addition, localized differences in phase retardation upon reflection of incident light from an opaque surface can be compared to the refractive index variations experienced with transmitted light specimens. For many applications in reflected light DIC, specimen details are frequently superimposed on a homogeneous phase background, a factor that dramatically benefits from contrast enhancement through optical staining (interference) techniques. With the thin transparent specimens that are optimal for imaging with transmitted light DIC, the range within which optical staining can be effectively utilized is considerably smaller (limited to a few fractions of a wavelength), rendering this technique useful only for thicker specimens.

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 with minimal interference from obscuring structures above and below the focal plane.

Figure 9 - Optical Sectioning in Reflected Light DIC Microscopy

In reflected light microscopy, the vertical illuminator aperture diaphragm plays a major role in defining image contrast and resolution. Reducing the aperture size increases the apparent depth of field and overall image sharpness while simultaneously producing enhanced contrast. However, if the diaphragm is closed too far, diffraction artifacts become apparent, image intensity is significantly reduced, and resolution is sacrificed. Often, the optimum aperture diaphragm setting is a compromise between accurately rendering specimen detail in sufficient contrast and retaining the resolution necessary to image minute features, while at the same time avoiding diffraction artifacts.

The series of high-magnification DIC images presented in Figure 9 illustrate three separate focal planes in the same viewfield of overlapping surface structures present on a typical integrated circuit. In brightfield or darkfield illumination, these structures are often observed merged together and can become quite confusing when attempting to image specific surface details. Figure 9(a) reveals several metal oxide terminals on the upper surface of the integrated circuit, including vias (miniature connections between vertical layers) and part of a bus line. Refocusing the microscope a few tenths of a micrometer deeper exposes numerous connections in the central region of the circuit (Figure 9(b)). Still farther into the circuitry, near the first layers applied above the pure silicon, are a series of metal oxide lines dotted with an ordered array of via connections (Figure 9(c)). The optical sectioning capability of reflected light DIC microscopy is clearly revealed by the ability to image specific focal planes on the surface of this complex integrated circuit.

Conclusions

An essential feature of both reflected and transmitted light differential interference contrast microscopy is that both of the sheared orthogonal wavefront components either pass through or reflect from the specimen, separated by only fractions of a micrometer (the shear distance), which is much less than the resolution of the objective. To the observer, it is not apparent that the resulting image visualized in the eyepieces is composed of these two superimposed components, because their separation is too minute to be resolved by the microscope. However, each point in the image is derived from two closely spaced and overlapping Airy disks originating from adjacent points on the specimen, and each disk has an intensity that corresponds to its respective optical path difference induced by the specimen. Image contrast is described as being differential because it is a function of the optical path gradient across the specimen surface, with steeper gradients producing greater contrast.

Primary candidates for observation in reflected light DIC microscopy include a wide variety of metallographic specimens, minerals, alloys, metals, semiconductors, glasses, polymers, and composites. The high resolution afforded by the technique has been employed to ascertain specimen details only a few nanometers in size. For example, spiral growth dislocation patterns in silicon carbide crystals that are only about 30-40 nanometers high can be imaged in high relief, while thin films approximately 200 nanometers thick have been successfully observed in monochromatic yellow sodium light. Phase transitions and recrystallization processes can be examined in reflected light DIC, as well as minute details on the surface of glasses and polymers.

Although largely a tool restricted to industrial applications, reflected light differential interference contrast microscopy is a powerful technique that has now been firmly established in the semiconductor manufacturing arena. The millions of computer chip components fabricated each year rely heavily on reflected light DIC to ensure quality control and help prevent failure of the circuits once they have been installed. Because of the countless hours spent by technicians examining integrated circuits, microscope manufacturers are now carefully turning their attention to ergonomic considerations in the design of new reflected light instruments. The result will undoubtedly be highly refined microscopes that produce excellent DIC images, while minimizing the discomfort and neuro-muscular disorders experienced by operators who must spend long periods repetitively examining identical specimens.

Contributing Authors

Chris Brandmaier - Industrial Microscope Division, Nikon Instruments, Inc., 1300 Walt Whitman Road, Melville, New York 11747.

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.