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.

Differential Interference Contrast

Interactive Tutorials

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.

The tutorial initializes with a beam of non-polarized white light impacting the polarizer from the left-hand side of the window. After passing through the polarizer (the gray disc), which has a transmission axis indicated by a long black wedge, the emerging linearly polarized light is oriented with the vibration direction oriented parallel to the fast axis of the quarter-wavelength retardation plate (represented by a black wedge on the light blue disc). In this configuration, the de Sénarmont compensator passes linearly polarized light, which is represented in the tutorial by a blue sine wave accompanied by an oscillating black arrow that traces the electric vector vibration orientation. When linear polarized light passes through the compensator, the retardation value is zero, as indicated in the yellow box positioned near the upper left-hand corner of the tutorial window. The entire compensator assembly can be rotated within the window by placing the mouse cursor on any component, and then dragging to model to a new position. For reference purposes, a schematic drawing is presented in Figure 1 that outlines the basic de Sénarmont compensator configuration motif for the input of linearly polarized light oriented at a 45-degree angle to the retardation plate fast axis.

The orientation of incident linearly polarized light with respect to the retardation (quarter-wavelength) plate can be altered by translating the Polarizer Rotation slider to either the right or left from its default central position. When the slider is moved to the left (negative values), between a range of 1 and 44 degrees, an increasing amount of light is passed through the slow axis of the retardation plate (indicated by a red sine wave exiting red wedges on the blue disc). The net result is the production of right-handed elliptically polarized light. The opposite effect occurs when the slider is moved to the right (positive values; left-handed elliptically polarized light). Elliptically polarized light is represented in the tutorial by a three-dimensional trajectory of the black arrow, which traces the vector sum of the individual electronic components from each wave (fast and slow).

When the Polarizer Rotation slider is moved either to the -45 or +45 degree position, light waves traveling through the fast and slow axes of the retardation plate have equal amplitudes (but with one wave retarded by 45 degrees), and the light emerging from the retardation plate is circularly polarized. In this case, the pathway scribed by the vector sum black arrow is circular. These effects are observed more clearly when the compensator is dragged to the front of the window and the waves are viewed end-on. As the Polarizer Rotation slider is translated, the retardation value of the complete de Sénarmont compensator is constantly updated and presented in the yellow box (near the upper left-hand corner of the tutorial).

The Fast and Slow radio buttons can be utilized to reverse the axes of the retardation plate (also reversing the handedness of the elliptically and circularly polarized light). Translation of the sine wave(s) can be halted by removing the check mark in the Translate Sine Wave checkbox. The Applet Speed slider controls the speed of the sine wave(s), while the Pause button will freeze the applet in the current configuration, but still enable rotation of the model through all three dimensions. Clicking the Reset button will return the tutorial to its initialization state.

Bias has been traditionally introduced into the differential interference contrast microscope by translating the objective Nomarski prism back and forth along the optical axis using a fine adjustment knob located at the end of the mounting frame (usually positioned in the microscope nosepiece housing or an intermediate tube). An alternative technique, 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 described above). At maximum extinction, 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, 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 45 degrees) with respect to the fast axis of the compensator, 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. However, when the polarizer transmission axis is rotated, wavefronts emerging from the compensator plate become elliptically polarized. Rotating the polarizer in one direction will produce right-handed elliptically polarized light, while rotating the polarizer in the other direction will alter the vector trajectory to generate a left-handed elliptical sweep.

Wavefront Relationships in de Sénarmont and Nomarski DIC

In differential interference contrast (DIC) microscopy, the spatial relationship and phase difference between ordinary and extraordinary wavefronts is governed either by the position of the objective prism (Nomarski DIC) or the relationship between the polarizer and a thin quartz retardation plate in a de Sénarmont design. This interactive tutorial explores the similarities and differences between the wavefront relationship in the two microscope configurations.

The tutorial initializes with a de Sénarmont DIC microscope optical train bearing a condenser Wollaston prism and an objective Nomarski prism appearing in the window. A beam of linearly polarized light emerges from the polarizer and becomes separated into two orthogonal components before entering the lower Wollaston prism (labeled the Condenser Prism) and being 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 Nomarski 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. Note that the raytrace paths are simplified schematic diagrams and not intended to accurately represent the actual refracted wavefront field passing through the optical system.

The microscope can be changed from a de Sénarmont to a traditional Nomarski configuration by toggling the Imaging Mode set of radio buttons (labeled de Sénarmont and Nomarski). In Nomarski imaging mode, the beam of linearly polarized light emerging from the polarizer first enters the lower Wollaston prism (labeled the Condenser Prism) before being separated into two orthogonal components and sheared at the quartz wedge interface. In de Sénarmont mode, the Polarizer Rotation Angle slider controls the relationship between wavefronts leaving the quarter wavelength retardation plate. The optical path difference between wavefronts is determined by the position of the polarizer, with respect to the retardation plate (prior to entry into a Wollaston or Nomarski prism), in de Sénarmont mode. However, in Nomarski mode, the Objective Prism Position slider is utilized to the create a similar optical path difference.

In order to operate the tutorial, use either the Objective Prism Position (Nomarski mode) or Polarizer Rotation Angle (de Sénarmont mode) slider to translate the objective prism laterally across the optical path of the microscope or rotate the polarizer, respectively. As the waveform relationship is altered, 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. The speed of wavefronts traveling through the virtual microscope can be increased or decreased using the Applet Speed slider.

Bias has been traditionally introduced into the differential interference contrast microscope by translating the objective Nomarski prism back and forth along the optical axis using a fine adjustment knob located at the end of the mounting frame (usually positioned in the microscope nosepiece housing or an intermediate tube). An alternative technique, 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). At maximum extinction, 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, 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) 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. 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, while rotating the polarizer in the other direction will alter the vector trajectory to generate a left-handed elliptical sweep.

When the orientation of the polarizer transmission axis reaches either plus or minus 45 degrees (equivalent to one-quarter wavelength 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 prism and become sheared. 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.

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. As previously discussed, in a properly configured microscope that is 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.

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.

Matthew 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.

When the orientation of the polarizer transmission axis reaches either plus or minus 45 degrees (equivalent to one-quarter wavelength 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 prism and become sheared. 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.

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 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.

Presented in Figure 2 is a series of digital images recorded in DIC using a bias retardation range of one-twentieth to a quarter wavelength in several intermediate steps. The specimen is a 15-micrometer section of fixed and mounted murine intestine that contains regions of fluctuating thickness. Rendition of specimen detail and the shadow-cast pseudo three-dimensional effects are the most pronounced at the lower bias retardation values (Figures 2(a) and 2(b)), and both contrast and definition of fine specimen detail deteriorate as bias retardation is increased (Figures 2(c) through 2(f)). At the highest bias retardation value (one-quarter wavelength; Figure 2(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, 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 2). 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.

Contributing Authors

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.

Optical Sectioning with de Sénarmont DIC Microscopy

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

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 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.

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 that the Nomarski prism components, analyzer, and de Sénarmont compensator be accurately aligned.

Figure 1 - Optical Sectioning in de Sénarmont DIC Microscopy

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 1. 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 1(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 1(b)). As the microscope is focused on the uppermost layer of the colonies in the mucilage (Figure 1(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.

Contributing Authors

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

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.

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

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.

The tutorial initializes with three-dimensional images of a polarizer and quarter-wavelength retardation plate (together comprising a de Sénarmont compensator), along with a fixed Wollaston prism, appearing in the window. From the left-hand side of the window, a beam of non-polarized white light is incident on the polarizer. After passing through the polarizer (the gray disc), which has a transmission axis indicated by a long black wedge, the emerging linearly polarized light is oriented with the vibration direction parallel to the fast axis of the quarter-wavelength retardation plate (represented by a black wedge on the light blue disc). In this configuration, the de Sénarmont compensator passes linearly polarized light, which is represented in the tutorial with a blue sine wave accompanied by an oscillating black arrow that traces the electric vector vibration orientation.

Linearly polarized light exiting the de Sénarmont compensator enters the Wollaston prism, and is separated into orthogonal (mutually perpendicular) components consisting of an ordinary and an extraordinary wave. These perpendicular wavefronts are sheared and exchange identities at the junction between the two quartz wedges in the Wollaston prism. The sheared orthogonal wavefronts also diverge at a very small angle (greatly exaggerated in the tutorial) upon encountering the wedge boundary. To enhance visualization, the entire optical train assembly can be rotated within the window by placing the mouse cursor on any component, and then dragging to model to a new position.

The orientation of incident linearly polarized light with respect to the retardation (quarter-wavelength) plate can be altered by translating the Polarizer Rotation slider to either the right or left from its default central position. When the slider is moved to the left (negative values), within a range of 1 and 44 degrees, an increasing amount of light is passed through the slow axis of the retardation plate (indicated by a red sine wave exiting red wedges on the blue disc). The net result is the production of right-handed elliptically polarized light. The opposite effect occurs when the slider is moved to the right (positive values; left-handed elliptically polarized light). Elliptically polarized light is represented in the tutorial by a three-dimensional trajectory of the black arrow, which traces the vector sum of the individual electronic components from each wave (fast and slow).

In order to operate the tutorial, introduce varying amounts of bias retardation into the optical train using the Polarizer Rotation slider, and then drag the model to different positions in the window to view the behavior of polarized wavefronts passing through the system. The sine wave(s) can be halted by removing the checkmark from the Translate Sine Wave check box, or the entire tutorial can be suspended in action by clicking on the Pause button. The speed of the sine wave can be increased or decreased with the Speed slider (the default setting is medium speed), and the tutorial can be re-initialized without reloading by using the Reset button.

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 1 - 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 orientation-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 1(b)), the fast axis of the retardation plate is aligned with the transmission axis of the polarizer. 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, 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 1(a) and 1(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 1(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 1(a)), while rotating the polarizer in the other direction will alter the vector trajectory to generate a left-handed elliptical sweep (illustrated in Figure 1(c)).

Figure 2 - 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 2). 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 2 for three settings of the compensator. All of the examples presented in Figure 2 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 (Figures 1(b) and 2(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 2(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 2(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 2(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 2(b)) 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.

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.

Matthew 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.

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 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.

The tutorial initializes with a cutaway model of a de Sénarmont compensator designed to fit a Nikon upright microscope appearing in the window. A beam of non-polarized light is incident on the normal plane of the polarizing element in the compensator, and passes through the linear polarizer before impacting on the quarter-wavelength plate at a 45-degree angle to the fast and slow axes. The polarized light, thus oriented, emerges from the compensator in the form of two orthogonal wavefronts that combine to produce right-handed circularly polarized light. In the tutorial, polarized light waves oriented parallel to the fast axis of the compensator (indicated by a red wedge on the plate) are represented as blue sine waves, while those polarized waves oriented parallel to the compensator slow axis (black wedge on the plate) are depicted as red sine waves.

The pathway of circularly polarized light exiting the compensator is calculated by the vector summation of the orthogonal wave components, and is illustrated in the tutorial as a black arrow that traces a circular arc around the periphery of the wavefronts. In all cases, the sine waves emerging from the de Sénarmont compensator are exaggerated in size to more clearly demonstrate their relationship to one another and to the compensator axes. The entire compensator assembly can be rotated within the tutorial window by placing the mouse cursor on any component, and then dragging the model to a new position.

In order to operate the tutorial, use the Polarizer Orientation slider to change the orientation of the polarizer with respect to the quarter-wavelength retardation plate in the compensator. As the slider is translated from the default position (+45 degrees), the phase relationship between orthoganol wavefronts is altered, and emerging polarized light changes from being circularly polarized to having varying degrees of elliptical polarization. These effects are most clearly observed when the top of the compensator model is dragged to the front of the window and the wavefronts are viewed end-on.

When the polarizer transmission axis becomes perfectly aligned (parallel) with the fast axis of the retardation plate (in this case, the Polarizer Orientation slider is set to zero degrees), only linear light emerges from the de Sénarmont compensator and no bias retardation is introduced into the optical system. Moving the slider further to the left (negative values) produces left-handed elliptically polarized light at polarizer orientations between 1 and 44 degrees, and circularly polarized light again at -45 degrees. The total amount of bias retardation introduced into the system is constantly updated as the Polarizer Orientation slider is translated, and the current value is presented in the yellow box positioned in the upper left-hand corner of the tutorial window.

Translation of the sine wave(s) can be halted by removing the check mark in the Translate Sine Wave checkbox. The Applet Speed slider controls the speed of the sine wave(s) passing through (and emerging from) the compensator, and should be used to slow the progression of wavefronts for observation. Clicking the Pause button will freeze the tutorial in the current configuration, but still enable rotation of the model through all three dimensions. Clicking the Reset button will return the tutorial to its initialization state.

In a majority of the traditional DIC microscope configurations, bias retardation is introduced by translating the objective Nomarski prism back and forth along the optical axis using a fine adjustment (micrometer) knob located at the end of the mounting frame (which is usually positioned in the microscope nosepiece housing or an intermediate tube). An alternate and more quantitative technique, termed the de Sénarmont DIC compensation method and now utilized by Nikon and other manufacturers, employs a quarter-wavelength retardation plate in fixed orientation between the polarizer and condenser prism. At maximum extinction, 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 an upright microscope or above the condenser for an inverted microscope. An alternative location for the de Sénarmont compensator, in microscopes equipped with the appropriate intermediate tube, is between the objective prism and the (rotatable) analyzer.

Figure 1 - de Sénarmont Compensator Wavefronts

In order to introduce bias retardation using the de Sénarmont compensator, the polarizer transmission axis is rotated (up to plus or minus 45 degrees) 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 Nomarski prism (as illustrated in Figure 1(b)). However, when the polarizer transmission axis is rotated, wavefronts emerging from the quarter-wavelength retardation plate in the de Sénarmont compensator become elliptically polarized (depending upon the orientation) as presented in Figures 1(a) and 1(c). Rotating the polarizer in one direction (positive) will produce right-handed elliptically polarized light, while rotating the polarizer in the other (negative) direction will alter the vector trajectory to generate a left-handed elliptical sweep.

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 de Sénarmont 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 retardation is introduced to the system when the wavefronts enter the Nomarski prism (and become sheared) located in the microscope condenser. 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.

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 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.

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.

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

DIC Microscope Configuration and Alignment

HomeLearnMicroscopy Resource CenterMicroscopy PrimerSpecialized Microscopy TechniquesdicDifferential 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 × 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.

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.

Fundamental Concepts in DIC Microscopy

HomeLearnMicroscopy Resource CenterMicroscopy PrimerSpecialized Microscopy TechniquesdicDifferential Interference Contrast - Fundamental Concepts

Living cells and other transparent, unstained specimens are often difficult to observe under traditional brightfield illumination using the full aperture and resolution of the microscope objective and condenser system. Phase contrast, first developed by Frits Zernike in the 1930s, is often employed to image these challenging specimens, but the technique suffers from halo artifacts, is restricted to very thin specimen preparations, and cannot take advantage of the full condenser and objective apertures.

The basic differential interference contrast (DIC) system, first devised by Francis Smith in 1955, is a modified polarized light microscope with two Wollaston prisms added, one to the front focal plane of the condenser and a second at the rear focal plane of the objective (see Figure 1). Several years later, Georges Nomarski, a Polish-born French physicist, modified the standard Wollaston prism configuration to enable these exceedingly thin optical components to be physically located away from the aperture conjugate planes.

The optical components required for differential interference contrast microscopy do not mask or otherwise obstruct the objective and condenser apertures (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 along 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.

Presented in Figure 1 is the typical differential interference contrast configuration for a modern transmitted light microscope also equipped for fluorescence illumination. The basic optical scheme closely resembles a traditional polarized light microscope retrofitted with specialized beamsplitting prisms. A polarizer and analyzer are inserted into the optical pathway before the condenser and after the objective, respectively. Several beamsplitting (modified Wollaston or Nomarski) prisms designed to accommodate objectives having differing focal lengths and aperture sizes are installed in the condenser turret assembly, while a single Nomarski prism (compatible with all objective specifications) resides in a slider frame positioned in the nosepiece. The relative optical orientation and sequential positioning of these auxiliary components is also indicated in the figure.

Unlike phase contrast, differential interference contrast converts gradients in specimen optical path length into amplitude differences that can be visualized as improved contrast in the resulting image. The specimen optical path difference is determined by the product of the refractive index difference (between the specimen and its surrounding medium) and the geometrical distance (thickness) traversed by a light beam between two points on the optical path. Images produced in differential interference contrast microscopy have a distinctive shadow-cast appearance, as if they were illuminated from a highly oblique light source originating from a single azimuth. Unfortunately, this effect, which often renders specimens in a pseudo three-dimensional relief, is frequently assumed by uninformed microscopists to be an indicator of actual topographical structure.

Differential interference contrast microscopy, unlike the situation with traditional double-beam interference instruments, is largely a qualitative rather than a quantitative technique. Specimens are sampled by two closely spaced partially coherent, but orthogonal, wavefronts separated by a distance that is slightly beneath the lateral resolution of the microscope. Because the sampling and reference beams both traverse a similar region of the specimen (and/or the surrounding medium), which is confined to a spatial separation distance of less than two micrometers, DIC will not yield accurate measurements of specimen refractive index or thickness. Instead, the technique is useful for determining the orientation of phase gradients and capitalizing on the full objective aperture to produce thin optical sections free of obscuring disturbances from specimen features positioned beyond the immediate focal plane.

The wave pairs employed in differential interference contrast are generated by the action of a birefringent beamsplitter (either a Wollaston or Nomarski compound prism) on a plane-polarized wavefront of coherent light originating from a tungsten filament and focused into the front focal plane of the microscope condenser (where the beamsplitter is positioned). When a pair of coherent light rays produced by the beamsplitter encounters a phase gradient, due to refractive index and/or thickness variations, each ray will become deformed and experience a slightly different optical path difference when traversing through the specimen. Upon emerging from the specimen, the rays will be unequal in phase. The difference in optical path is translated by the DIC microscope into a change in amplitude in the final image observed in the eyepieces. However, from simply examining the image, it is impossible to determine whether the phase gradient in the specimen occurs because of differences in refractive index or thickness (or both). This uncertainty is due to the fact that optical path difference is derived from the product of refractive index and thickness, and lacking independent information about either quantity, the origin of the difference cannot be ascertained.

After 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 passed 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 2. The specimen presented in Figure 2(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) verses the diameter of the erythrocyte along the shear axis (the abscissa) is shown in Figure 2(b). Note that the optical path profile reflects the thin center and thick edges exhibited by human red 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 2(b)) when added to a constant, is presented in Figure 2(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.

Differential Interference Contrast Optical Configuration

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 Figure 1). 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 - Inserted into the optical pathway between the microscope light port (or anywhere after the illumination source collector lens) and the condenser lens assembly (see Figures 1 and 3), 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, as will be discussed subsequently.

- Condenser Wollaston or Nomarski Prism - 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 iris diaphragm aperture, as illustrated in Figure 3. 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.

- Objective Nomarski Prism - Positioned behind the objective (Figure 3), 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 - 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 (Figure 3). 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).

When the Wollaston and/or Nomarski prisms are removed from the differential interference contrast microscope optical pathway, the optical configuration is equivalent to standard polarizing instrument adjusted for maximum extinction (crossed polarizers). Because the DIC technique relies on plane-polarized light, birefringent specimens or strained optical components can interfere with image intensity by producing unfocused bright regions on an otherwise dark (or black) background. For this reason, DIC microscope configurations should employ strain-free objectives and (preferably) condenser lens elements. Standard microscope objectives often contain stress signatures in the glass arising from tight lens mounts, occlusions, and birefringent inhomogeneities in the lenses. These defects often result in lowered contrast levels, which can have serious consequences to the fidelity of the final image. In addition, the close proximity of separated wavefronts (slightly less than the diffraction-limited resolution) requires high-precision specifications for microscope objectives, especially at large magnifications, in order to achieve the full resolution potential of the technique.

Presented in Figure 3 is an idealized schematic diagram of the major components and light pathways through a typical DIC microscope optical train. Coherent wavefronts emitted from localized regions of the lamp filament pass through the polarizer to form linearly polarized light oriented parallel to the axis indicated by the double arrow adjacent to the polarizing element (45 degrees with respect to the plane of the page). The polarized wavefronts converge on the condenser front focal plane where a Wollaston combination prism is located.

After being sheared by the prism (discussed below), the resulting orthogonal or mutually perpendicular wavefronts are illustrated as a series of red double arrows (wavefront parallel to the page) and blue dots (wavefronts perpendicular to the page). Once they have traversed optical path gradients in the specimen, the wavefronts are gathered by the objective and converge at the rear focal plane, where a second Wollaston prism is positioned. The recombined wavefronts then pass through a second polarizer (the analyzer), which is oriented with a transmission axis indicated by the black double arrow to the left of the polarizing element (90 degrees with respect to the sub-condenser polarizer). Note that the condenser prism is imaged onto the objective prism in Figure 3, 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 and parallel to the page, as discussed below) produces a wavefront mismatch that is uniform across the microscope aperture.

Wollaston and Nomarski Prisms

Birefringent Wollaston and/or Nomarski prisms are inserted in the optical pathway with their shear axis oriented at a 45-degree angle (northwest to southeast) to the polarizer and analyzer. The prisms are composed of two precisely ground and polished wedge-shaped slabs produced from high-grade optical quartz, a uniaxial birefringent crystal. Two quartz wedges having perpendicular orientations of the optical axis must be fabricated to produce a single Wollaston (or Nomarski) prism. The wedges are cemented together at the hypotenuse to generate an optically anisotropic compound plate where the crystallographic optical axis of the first wedge is perpendicular to the optical axis of the second wedge. Incident linearly-polarized wavefronts that enter a prism (oriented with the optical axis at a 45-degree angle to the polarized light) in the condenser aperture are divided into two separate orthogonal waves, termed the ordinary and extraordinary wave.

The mutually perpendicular extraordinary and ordinary component wavefronts are coherent, have identical amplitudes (70.7 percent of the original polarized wave), and travel in the same direction through the lower half of the Wollaston prism. However, the waves propagate at different velocities, which are defined by the dielectric properties along the slow and fast axes of the lower birefringent quartz crystalline wedge. The ordinary wave proceeds through the prism along the fast axis (having a lower refractive index), while the extraordinary ray travels through the slower axis, which has a higher refractive index. In quartz, the refractive index difference between the fast and slow axes is approximately 0.6 percent, and the fast axis is oriented perpendicular to the crystallographic axis of the wedge. Therefore, the ordinary wave traverses a quartz wedge section perpendicular to the optical axis, while the extraordinary wave is oriented parallel to this axis.

An angular splitting or shear of the wavefronts occurs at the refractive index junction between the cemented quartz wedges, 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 (Figure 4). One wavefront (the ordinary) propagates from a medium of low refractive index into a second medium (the upper wedge) of higher refractive index and is bent toward the normal (perpendicular to the wedge optical axis) according to Snell's law. The second wavefront (the extraordinary) leaves a medium of high refractive index and enters a second medium of lower refractive index, bending the wavefront away from the normal, but at the same angle as the first wavefront.

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 (crystallographic) axis of the lower quartz wedge section (as illustrated in Figure 4). As a result, one of the polarized wavefronts entering a Wollaston prism will be oriented parallel to the direction of the shear axis, while the other is oriented perpendicular to this axis. The shear angle is determined by the design of the prism components (quartz wedge angles, which are less than a degree of arc) and cannot be adjusted in the microscope. However, the shear distance is so minute (usually less than a micrometer) that no observable beam separation occurs in light emerging from the prism.

During their journey through the lower quartz wedge in a Wollaston prism, the ordinary and extraordinary wavefronts experience different refractive indexes, as discussed above. As a result, the ordinary wavefront propagates through the crystal at a higher velocity than does the extraordinary wavefront. When the wavefronts exchange identities at the interface between the lower and upper quartz wedge, the ordinary wavefront becomes the extraordinary wavefront, and vice versa. In addition, the wavefronts undergo a phase shift in the lower half of the prism (due to refractive index differences) that is exactly compensated in the upper half when the geometric paths through the lower and upper half of the Wollaston prism are identical (Figure 4(b)). Wavefronts traversing the prism away from the center experience a longer journey through either the lower prism wedge before being sheared (Figure 4(c)), or the upper wedge after being sheared (Figure 4(a)), before exiting. The extended distance traveled through a single prism wedge by the wavefronts ultimately enables one of the waves (either the ordinary (Figure 4(a)) or extraordinary (Figure 4(c)) to arrive at the quartz-air interface ahead of the other. Across the face of the prism, a constant phase shift per unit length occurs in the direction of shear that is equal, but opposite, for the ordinary and extraordinary wavefronts (Figure 4(a) and 4(c)). 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.

If the wavefront incident on a Wollaston prism is polarized in an orientation parallel to the shear axis, then orthogonal splitting of the wavefront will not occur and plane-polarized light will emerge from the prism. Likewise, if the incident polarized wavefront is oriented perpendicular to the prism shear axis, it too will emerge from the prism unchanged with respect to orientation. The ideal situation (and necessary for differential interference contrast microscopy) occurs when the incident polarized wavefront is oriented at a 45-degree angle to the shear axis of the prism. The electric vector of linearly polarized light entering from this angle is bisected into two component vectors, each vibrating in the plane of the fast or slow axis of the quartz crystal and having the root-mean-square (70.7 percent) amplitude of the original wavefront. Both Wollaston and Nomarski prisms exhibit orientation-dependent properties. A collimated linearly polarized light beam entering the opposite side of the prism (this time from the top) at a 45-degree angle will also produce orthogonal extraordinary and ordinary wavefronts. However, the polarization of the waves will be reversed.

When a Wollaston or Nomarski 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 (illustrated in Figure 5). These patterns result from interference between the obliquely pitched ordinary and extraordinary wavefronts emerging across the face of the prism. To the left and right of the central dark interference fringe, the peripheral fringes display increasing orders of the classical polarization interference color spectrum. Prism wedges designed for objectives having different focal lengths and numerical apertures are cut at increasingly shallow angles (as magnification and numerical aperture increase) to yield narrower interference fringe banding. This concept is illustrated on the right-hand side of Figure 5 for a series of fixed Nomarski prisms designed for successively higher objective magnifications (indicated in the figure).

If a first-order compensator (red plate) is added to the crossed polarizer sandwich in a diagonal position (not illustrated in Figure 5), the black fringe is replaced with interference colors showing subtraction (yellow) on one side and addition (blue) on the other side of the original dark fringe position. Adding a second Wollaston or Nomarski prism on top of the first will compensate the phase shifts (and resulting interference fringes) of the first prism across the entire length, resulting in extinction (illustrated in Figure 5; note that this effect can only be observed if the two prisms employed for the experiment have the same shear angle). By translating one of the prisms laterally with respect to the other, a uniform bias, or change in path length, will be introduced and can be observed through the sandwich (Figure 5). Sliding the prism in one direction will darken and then lighten the prisms, while sliding it in the other direction will produce a series of uniform interference colors (starting with first-order yellow).

Interference fringes observed in a Nomarski prism sandwiched between two polarizers appear to float in space a few millimeters above the prism. However, when the same fringes are viewed using a Wollaston prism, they appear to lie inside the prism. The location of the interference fringes for either a Nomarski and Wollaston prism is termed the interference plane. Because the interference plane in a conventional Wollaston prism is positioned in the central portion of the prism, at approximately the centerline between the wedges (Figure 6), it is difficult to adapt a Wollaston prism for use with standard microscope objectives. This problem arises because the interference plane of the prism must coincide and overlap with the rear focal plane (also termed the diffraction plane) of the objective, which often lies below the thread mount inside a glass lens element.

Most manufacturers circumvent the objective aperture clearance problem by employing Nomarski (occasionally termed modified Wollaston) prisms both for beam shearing and recombination duty in the condenser and objective focal planes, respectively. Due to a specialized design, as discussed below, Nomarski prisms have an interference plane that is displaced to a site several millimeters outside the prism, rather than spanning the wedge elements as in the traditional Wollaston design. Nomarski prisms do not require being physically located in the objective or condenser focal plane, but can be positioned some distance away. Although Nomarski prisms do not produce better contrast, they avoid the potential problem of interference fringes becoming visible in the viewfield. It should be noted that Leica Microsystems once produced a popular microscope known as the Smith T differential interference contrast system, which incorporated standard Wollaston prisms into specially designed objectives. However, this design strategy is the rare exception, rather than the rule.

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 6. This effect occurs because shear now takes place at the air-quartz interface of the lower wedge, 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.

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.

It is important to remember that aligning the microscope for Köhler illumination is a critical and requisite step to ensure correct positioning of the Nomarski prism interference planes for coincidence with the conjugate aperture planes of the condenser and objective. The central, or zeroth-order interference fringe, which is observed when a Nomarski prism is placed between crossed polarizers (as described above), can be used to determine the correct orientation of the prisms during alignment of the microscope.

DIC Wavefront Relationships and Image Formation

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 differences 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 7(a) and 7(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 7(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 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, and simple, darkfield illumination technique.

These concepts are depicted graphically in Figure 8, which presents phase relationships between the sheared wavefronts after passing through a phase specimen (having higher refractive index than the surrounding medium), and their corresponding amplitude (or intensity) profile at the image plane. Mutually perpendicular wavefronts (labeled Σ(1) and Σ(2); see Figure 8(a)) passing through the specimen are distorted and display localized regions of phase retardation (termed differential phase retardations). The objective Nomarski prism recombines the wavefronts by canceling the angular wave shear introduced by the condenser prism, generating a lateral displacement of the wavefront deformation in the process. The distorted wavefront profiles (for both the extraordinary and ordinary components) reconstructed in the image plane along the shear axis are illustrated in Figure 8(a) when the dual prism instrument configuration has been adjusted to maximum extinction. Phase retardation (φ) introduced to the wavefronts by the specimen is indicated on the ordinate (measured in nanometers), while the width of the dip along the shear axis (x) represents the magnified specimen diameter (in this case, a single droplet of oil).

After the phase-retarded wavefronts in Figure 8(a) have been transmitted through the analyzer and are reunited by constructive and destructive interference at the image plane, the resulting intensity distribution can be represented as an amplitude plot along the shear axis (Figure 8(b)). For the symmetrical oil drop specimen considered in this example, the amplitude plot as a function of distance across the shear axis produces a dark central cavity flanked on either side by bright regions (see Figure 8(b)). A digital image of the actual specimen observed in the microscope (Figure 8(c)) reveals spherical microdomains having bright edges superimposed on a black background and a dark interference fringe banding the center. The images presented in Figure 8 were recorded on an upright microscope configured with a DIC optical system. Inverted tissue culture microscopes produce the same basic result, however, the central interference fringes bisecting the hemispherical oil droplets will be oriented perpendicular to those illustrated in Figure 8(c).

Introduction of Bias Retardation

In a differential interference contrast microscope adjusted for maximum extinction, the viewfield is rendered with a dark, almost black, background and exhibits very high sensitivity to specimen regions having both increasing and decreasing phase gradients. As illustrated in Figure 8(c), some specimen detail is obscured or very difficult to resolve in this configuration, and the zeroth-order interference fringe can often be observed traversing highlighted features. In practice, the objective Nomarski prism is offset laterally along the shear axis to uniformly shift the relative phase displacement of the ordinary and extraordinary wavefronts passing through the specimen. Thus, the polarization vector orientation of light emerging from the objective prism can be adjusted from linear through various degrees of elliptical and even to circular. Shifting the phase displacement of the ordinary wavefront with respect to the extraordinary wavefront through translation of the objective prism is often termed introduction of bias retardation in DIC microscopy.

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.

Intensity gradients introduced to the specimen by altering the bias retardation 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 (see Figure 8(f)). Note that the gradients illustrated in Figure 8(f) were recorded with an upright microscope. Inverted microscopes produce intensity gradients that are oriented perpendicular to those observed in upright microscopes. Shifting the prism in one direction or another will affect the interference fringe offset and vary the phase relationship (retarded or advanced) between the ordinary and extraordinary wavefronts, thus reversing the shadow-cast orientation in the specimen.

The net result of introducing bias retardation is to render the specimen image in pseudo three-dimensional relief where regions of increasing optical path difference (sloping phase gradients) appear much brighter (or darker), and those exhibiting decreasing path length appear in reverse. Specimen features appear similar to elevated plateaus or sunken depressions depending on the phase gradient orientation, which is a distinguishing feature of differential interference contrast. However, the three-dimensional appearance only corresponds to phase gradients and should not be confused with the actual specimen geometry (as is too often the case). In some situations, real topographical features are also sites of changing phase gradients, but in the absence of information obtained as the result of independent investigations, this fact should not be assumed.

Introduction of bias retardation in differential interference contrast microscopy is illustrated in Figure 8(d) through Figure 8(f) for a phase specimen consisting of several hemispherical oil droplets. When the microscope is adjusted for maximum extinction, the ordinary and extraordinary wavefronts display a phase shift along the shear axis, but do not exhibit a phase difference in regions corresponding to the background (Figure 8(a)). Addition of bias retardation by translation of the objective prism shifts the relative phase of one wavefront with respect to the other (Figure 8(d)), but the wavefront shear remains the same. Upon interference at the image plane, the resulting amplitude (or intensity) plot, as a function of shear distance (Figure 8(e)), displays a bright edge region on one side of the oil droplet and a dark region on the opposite side. When observed in the microscope, the specimen exhibits a shadow-cast appearance as if it were being illuminated from a highly oblique angle (see Figure 8(f)). In order to observe the difference between maximum extinction and addition of bias retardation, compare the specimen images presented in Figure 8(c) and (f). The shadow orientation, which is dependent on the shear axis (double-headed arrows in Figure 8(c) and (f)), can be reversed by translating the objective prism by the same amount in the opposite direction.

Bias has been traditionally introduced into the differential interference contrast microscope by translating the objective Nomarski prism back and forth along the optical axis using a fine adjustment knob located at the end of the mounting frame (usually positioned in the microscope nosepiece housing or an intermediate tube). An alternative technique, 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). At maximum extinction, 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, 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) 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. 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, while rotating the polarizer in the other direction will alter the vector trajectory to generate a left-handed elliptical sweep.

When the orientation of the polarizer transmission axis reaches either plus or minus 45 degrees (equivalent to one-quarter wavelength 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 prism and become sheared. 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.

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. As previously discussed, in a properly configured microscope that is 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.

Presented in Figure 9 is a series of digital images recorded in DIC using a bias retardation range of one-twentieth to a quarter wavelength in several intermediate steps. The specimen is a 15-micrometer section of fixed and mounted murine intestine that contains regions of fluctuating thickness. Rendition of specimen detail and the shadow-cast pseudo three-dimensional effects are the most pronounced at the lower bias retardation values (Figure 9(a) and 9(b)), but contrast and definition of fine specimen detail both deteriorate as bias retardation is increased (Figure 9(c) through 9(f)). At the highest bias retardation value (one-quarter wavelength; Figure 9(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, 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 9). 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 DIC Microscopy

Bias retardation between the ordinary and extraordinary wavefronts in differential interference contrast can also 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 optical phase 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 10 are several transparent specimens that have been optically stained and rendered in pseudo three-dimensional relief through DIC optical techniques. Figure 10(a) depicts projections at the edge of a ctenoid fish scale, while the mouth of a canine hookworm (Ancylostoma caninum) is featured in Figure 10(b). Colorful wing scales of the Great Leopard moth (Ecpantheria scribonia) are presented in Figure 10(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. As discussed above, 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 microscopy, de Sénarmont compensators are often utilized to optimize contrast in specimen detail that lies below the resolution limit of the microscope.

Interpretation of DIC Images

Among the most notable and well-recognized aspects of images obtained with differential interference contrast microscopy is the high degree of visible relief, manifested through a shadow-cast effect that bestows a pseudo three-dimensional realism. In general, specimens appear as through they were illuminated from a low angle with a highly oblique light source, reminiscent of the results obtained with traditional oblique illumination or Hoffman modulation contrast. In caution, however, images should always be interpreted with the understanding that shadows and highlights in the shadow-cast renditions only indicate the sign and slope orientation of phase or optical path gradients and do not necessarily reveal accurate geometrical or topographical parameters.

From observing the shadow-cast orientation present in almost every image produced through bias retardation, the direction of optical shear is obvious and can be precisely defined as the axis connecting regions displaying the highest and lowest intensity values. Another consideration that should be taken into account is the relationship between the specimen and its surrounding medium, because shadow directions are often reversed for specimen details that possess either a higher or lower refractive index than their surroundings. As a result, dense subcellular particles, such as nuclei, nucleoli, mitochondria, filaments, metaphase chromosomes, and lysosomes usually display the appearance of raised elevations (hilltops), while lower refractive index inclusions (for example, pinocytotic vesicles, aqueous vacuoles, and lipid droplets) appear to be sunken depressions (craters).

The level of contrast bestowed on specimen phase gradients (and the degree of pseudo three-dimensionality) by differential interference contrast is a function of the amount of bias retardation introduced into the optical system by translating the Nomarski prism or rotating the polarizer in a de Sénarmont compensator. Because the shear axis is fixed by Nomarski and Wollaston prism design and other constrains involved in wavefront orientation for differential interference contrast, 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 relocating the objective prism from one side of the microscope optical axis to the other (shifting the bias retardation from negative to positive, or vice versa). This operation can also be accomplished by rotating the polarizer to the corresponding negative value on a de Sénarmont compensator. 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 (designed primarily for polarized light microscopy).

In differential interference contrast images, shadow and highlight intensity 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. When a specimen having spherical geometry (Figure 8(f)) is examined from several azimuths for contrast at the edges bordering the background, it is discovered that contrast is minimal in the regions perpendicular to the shear axis. In fact, contrast differences between the specimen and background gradually decrease until they reach a minimum value in the direction defining the axis of the zeroth-order interference fringe (perpendicular to the shear axis). In Figures 8(c) and 8(f), the specimen regions exhibiting the lowest level of contrast (indicated by an unlabeled white arrow) are the edges in the central area of the oil droplets lying exactly perpendicular to the shear axis. In order to verify this concept, compare an edge where the interference fringe meets the background in the image recorded at maximum extinction (Figure 8(c)) to the corresponding region in the image generated after introduction of bias retardation (Figure 8(f)). In the areas marked by the unlabeled white arrow, the ordinary and extraordinary wavefront distortions have aligned profiles, which are subtracted at the analyzer to cancel residual retardation and yield an intensity value that exactly matches the background.

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 approximately 30 nanometers). However, thicker specimens often require higher bias settings (up to one-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 retardation setting is usually a compromise.

Azimuthal effects of orientation phenomena in differential interference contrast for several specimens are presented in Figure 11. In all cases, the shear axis is directed northwest to southeast, but is not indicated on the individual digital images. Figure 11(a) and 11(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 11(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 fresh water Daphnia (water flea) is presented in Figure 11(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 11(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 11(e)). Rotating the specimen to bring the spines parallel to the shear axis (Figure 11(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.

Optical Sectioning

The ability to image a specimen in differential interference contrast with large condenser and objective numerical apertures enables the creation of remarkably shallow optical sections from a focused image. 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 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 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 12. The cell is approximately 3 micrometers thick near the nucleus, and depth of field for the configuration utilized to obtain the images in Figure 12 was approximately 0.5 micrometers. Bacteria are clearly visible on the upper surface of the cell (Figure 12(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 12(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 12(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.

Conclusions

Differential interference contrast microscopy is basically 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. In fact, each point in the specimen is represented by two overlapping Airy disks in the final image, one being brighter and the other darker than the background. The basic microscope system, first devised by Francis Smith 1955, is a modified polarized light microscope with two Wollaston prisms added, one to the front focal plane of the condenser and the second at the rear focal plane of the objective.

Later modifications, suggested by Georges Nomarski, enabled the prisms to be physically located away from the optical aperture conjugate planes. The condenser prism converts each wavefront illuminating the specimen into two slightly displaced, parallel beams that are orthogonally polarized relative to each other, while the objective prism serves to recombine the beams. The combination of these two prisms, which are mirrored onto one another by the optical system, is a key feature in the ability of differential interference contrast to form sharp images at high numerical aperture.

A phase difference is introduced into the two orthogonal wavefronts by a gradient in the geometric path length or refractive index of the specimen, which results in elliptical polarization for the recombined beam that exits the objective Wollaston prism. 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. Thus, optimum contrast, field brightness, and sensitivity can be gained by simple rotation of a control knob.

The resulting DIC images possess a shadow-cast appearance that effectively display the gradient of optical paths for both low and high spatial frequencies. Those specimen regions where optical paths increase along a reference direction appear brighter (or darker), while the regions where path differences decrease appear in reverse contrast. Steeper gradients in optical path difference result in greater contrast. A wide variety of specimens are good candidates for imaging with differential interference contrast, including very thin filaments or sharp interfaces, which produce good contrast even when their diameter falls below the resolution limit of the optical system.

Among the chief imaging advantages of differential interference contrast microscopy is that, unlike darkfield or phase contrast, the image of smaller specimen features is not obscured by adjoining regions having large optical 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, minutes appendages to living cells or dynamic inclusions and moving organelles inside a cell), is a significant improvement over traditional phase contrast 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.

Contrast in DIC microscopy is directional, exhibiting a maximum along the axis of shear and a minimum in the orthogonal direction. The direction of Airy disk separation (averaging a peak-to-peak separation of one-half to two-thirds the disk radius) coincides with the shear axis of the microscope, which is the direction of maximum contrast. As a result, the DIC contrast transfer function (CTF) is also directional along the shear axis. The lateral displacement between two wavefronts passing through the specimen is approximately half the resolution limit of the objective. This small degree of shear, induced by the Wollaston or Nomarski prism, is similar in action to a high pass filter for the contrast of spatial frequency detail in the specimen. The corresponding modulation transfer function (MTF) closely follows that observed in brightfield illumination for high spatial frequencies, but displays a steep decline for specimen features exceeding several micrometers in size (lower spatial frequencies).

Specimens suitable for observation in DIC include fluid smears, living cell cultures, blood cells, subcellular organelles, unstained tissues, chromosomes, protozoa, embryos, diatoms, polymers, replicas, and relatively thick or ultra thin microtome sections. Additional information can be obtained by examining amplitude and mixed phase-amplitude specimens, such as naturally pigmented protists, algae, and lightly stained histological specimens. The technique is often employed in combination with fluorescence microscopy to reveal the cellular morphology associated with fluorescent regions. When coupled to enhanced video techniques (termed VEC-DIC for video-enhanced contrast differential interference contrast), DIC can be utilized to produce images of structures having dimensions below the optical resolution 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.

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.

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.