Differential Interference Contrast - Fundamental Concepts (2023)

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 apertures. condenser totals and the target.

The basic differential interference contrast (DIC), first developed by Francis Smith in 1955, is a modified polarized light microscope with two Wollaston prisms added, one in the front focal plane of the condenser and a second in 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 allow these extremely thin optical components to be physically located far from the conjugate planes of aperture.

The optical components required for differential interference contrast microscopy do not mask or obstruct the objective and condenser apertures (as in Hoffman or phase modulation contrast), allowing 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. Additionally, differential interference contrast produces an image that can be easily manipulated using video and digital imaging techniques to further enhance contrast.

The typical differential interference contrast setup for a modern transmitted light microscope also equipped for fluorescence illumination is shown in Figure 1. The basic optical scheme resembles a traditional polarized light microscope equipped with specialized beam splitting prisms. A polarizer and analyzer are inserted in the optical path before the condenser and after the objective, respectively. Multiple beam-splitting prisms (Wollaston or modified Nomarski) designed to accommodate lenses with different focal lengths and aperture sizes are mounted in the condenser turret assembly, while a single Nomarski prism (compatible with all lens specifications) resides in one Sliding frame attached to the nose piece. The relative optical orientation and sequential positioning of these auxiliary components are also indicated in the figure.

Unlike phase contrast, differential interference contrast converts gradients in the optical path length of the sample into amplitude differences that can be displayed as enhanced contrast in the resulting image. The sample optical path difference is determined by the product of the refractive index difference (between the specimen and its surrounding medium) and the geometric distance (thickness) traveled by a beam of light between two points on the path. Images produced in differential interference contrast microscopy have a distinct shadow-cast appearance, as if illuminated by a highly oblique light source originating from a single azimuth. Unfortunately, uninformed microscopists often assume that this effect, which often turns specimens into a three-dimensional pseudo-relief, is an indicator of real topographical structure.

Differential interference contrast microscopy, unlike the situation with traditional dual-beam interference instruments, is largely a qualitative rather than a quantitative technique. Samples are sampled by two closely spaced partially coherent but orthogonal wavefronts separated by a distance slightly below the lateral resolution of the microscope. Because hesamplingyreferenceboth rays pass through a similar region of the sample (and/or surrounding medium), which is confined to a spatial separation distance of less than two micrometers, DIC will not produce accurate measurements of refractive index or sample thickness. Instead, the technique is useful for determining the orientation of phase gradients and taking advantage of the objective's full aperture to produce undisturbed thin optical sections that obscure sample features located beyond the immediate focal plane.

The wave pairs used in differential interference contrast are generated by the action of a birefringent beam splitter (a Wollaston or Nomarski composite prism) on a plane-polarized wavefront of coherent light originating from a tungsten filament and focused in the plane. front focal point of the microscope. condenser (where the beam splitter is placed). When a pair of coherent light rays produced by the beam splitter encounter a phase gradient, due to refractive index and/or thickness variations, each ray will be deformed and experience a slightly different optical path difference as it passes through the sample. As they emerge from the specimen, the rays will be out of phase. The difference in the optical path is translated by the DIC microscope into an amplitude change in the final image seen through the eyepieces. However, simply by examining the image, it is impossible to determine whether the phase gradient in the sample is due to differences in refractive index or thickness (or both). This uncertainty is due to the fact that the optical path difference is derived from the product of the refractive index and the thickness and, in the absence of independent information about any quantity, the source of the difference cannot be determined.

After the wavefronts generated by the beam-splitting prism pass through a sample phase gradient, they are recombined through differential interference from a second prism and an analyzer (another polarizer) to produce a high-contrast gradient representation. Both differential interference contrast and phase contrast are based on sample phase differences between the sample and reference beams to produce a variable amplitude based image. Phase contrast translates image amplitude information from the phase variations displayed between light waves diffracted by the sample and a reference beam passing through the condenser ring, sample, and phase plate. However, the DIC image corresponds to the first mathematical derivative, rather than the magnitude, of the gradient profile obtained from the optical path difference of the sample.

The relationship between light path gradients and intensity profiles in DIC microscopy is illustrated in Figure 2. The specimen shown in Figure 2(a) is a donut-shaped human erythrocyte imaged at high magnification in differential interference contrast with the shear axis indicated by a double-headed arrow (northwest to southeast). Figure 2(b) shows a plot of the difference in light path (the ordinate) versus the red blood cell diameter along the slice axis (the abscissa). Note that the light path profile reflects the thin center and thick edges that human red blood cells exhibit. An intensity sweep across the differential interference contrast image is shown in Figure 2(c) which corresponds very closely to the first derivative of the optical path difference curve (Figure 2(b)) when added to a constant. The positive and negative slopes in the RBC lightpath profile generate regions of greater and lesser amplitude, respectively, in the first derived scan and in the corresponding differential interference contrast image. The regions of the lightpath difference profile that show no change in slope have the same intensity as the background and correspond to the baseline on the plot of the first derivative of the lightpath difference.

Differential interference contrast optical configuration

Strategic placement of precisely matched optics at (or near) conjugate planes and other specific locations within the microscope is essential to the setup scheme for differential interference contrast (see Figure 1). All major manufacturers offer high quality, precision DIC optical accessories, usually sold in kits, for their upright and inverted research microscopes. In general, only four basic components are needed to set up a standard laboratory or research brightfield microscope for observation in differential interference contrast:

• linear polarizer- Inserted in the light path between the microscope's light port (or anywhere past the light source's collecting lens) and the condenser lens assembly (see Figures 1 and 3), this component is designed to produce plane polarized light required for the interference image. The transmission axis from the plane of vibration to the electric vector component is oriented in an east-west direction (right to left when facing the microscope), typical of a standard polarized light microscope. Some differential interference contrast designs incorporate a rotating polarizer combined with a quarter-wavelength delay plate at this microscope position. Together, these components are calledof Senarmontcompensator and are designed to provide finer control for adjusting image contrast, as discussed later.
• Wollaston Condenser or Nomarski Prism- To separate the polarized light emanating from the polarizer into two components, a specialized beam-splitter prism (often referred to as acondenser prism) is placed close to the conjugate focal plane of the condenser iris diaphragm aperture, as illustrated in Figure 3. Incident wavefronts of plane-polarized (orcut) into mutually perpendicular (orthogonal) polarized components (calledcommonyextraordinarywave fronts) by the prism of Wollaston or Nomarski.
• Nomarski objective prism- Positioned behind the objective (Figure 3), either in an adjustable sliding frame or in a fixed mount, a second beam-splitter prism is used to recombine the wavefronts clipped in the conjugate plane of the rear aperture of the objective. This component, which is a critical element for interference and imaging, is also calledtarget 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 the optically matched microscope aperture planes.
• analyzer- A second linear polarizer is installed behind the objective prism, usually in an intermediate tube between the microscope nosepiece and the observation tubes (eyepiece). calledanalyzer, this polarizing element is placed in the light path before the tube lens (for infinitely corrected microscopes) and the 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. Circular and elliptically polarized light components arriving from the objective prism pass through the analyzer and are subsequently interfered to generate the DIC image in the intermediate imaging plane of the microscope (fixed eye slit or projection camera system with lens aperture).

When the Wollaston and/or Nomarski prisms are removed from the light path of the differential interference contrast microscope, the optical setup is equivalent to the standard polarizing instrument set to maximum extinction (crossed polarizers). As the DIC technique is based on plane polarized light, birefringent specimens or strained optics can interfere with image intensity by producing out-of-focus bright regions on a dark (or black) background. For this reason, DIC microscope setups should employ strain-free objectives and (preferably) condensed lens elements. Standard microscope objectives often contain signs of stress on the glass from tight lens mounts, occlusions, and birefringent lens inhomogeneity. These defects often result in lower contrast levels, which can have serious consequences for final image fidelity. Furthermore, the proximity of the separate wavefronts (slightly less than diffraction-limited resolution) requires high-precision specifications for microscope objectives, especially at high magnifications, to achieve the technique's full resolution potential.

An idealized schematic diagram of the major components and light paths through a typical DIC microscope optical train is shown in Figure 3. 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 from the plane of the page). The polarized wavefronts converge at the front focal plane of the condenser where a Wollaston combination prism is located.

After being cut by the prism (discussed below), the resulting orthogonal or mutually perpendicular wavefronts are illustrated as a series of double red arrows (wavefront parallel to the page) and blue dots (wavefront perpendicular to the page). After they have traversed the optical path gradients in the sample, the objective brings the wavefronts together and they converge to the back focal plane, where a second Wollaston prism is placed. The recombined wavefronts then pass through a second polarizer (the analyzer), which is oriented with a transmission axis indicated by the double black arrow to the left of the polarizing element (90 degrees to the subcapacitor's polarizer). Note that the condenser prism is reflected by the objective prism in Figure 3, so the wavefront clipping equalizes at all points along the surface of the prisms (which are inverted with respect to each other). Translating either prism along the slice axis (perpendicular to the microscope optical axis and parallel to the page, as explained below) produces a wavefront mismatch that is uniform across the microscope aperture.

Wollaston and Nomarski prisms

Wollaston and/or Nomarski birefringent prisms are inserted into the light path with their cut axis oriented at a 45 degree angle (northwest to southeast) to the polarizer and analyzer. The prisms are composed of two precision-ground and polished wedge-shaped plates made from high-quality optical quartz, a uniaxial birefringent crystal. Two quartz wedges with orientations perpendicular to 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 composite plate where the crystallographic optical axis of the first wedge is perpendicular to the optical axis of the second wedge. Incident linearly polarized wavefronts entering a prism (oriented with the optical axis at a 45-degree angle to the polarized light) at the condenser aperture split into two separate orthogonal waves, calledcommonyextraordinaryola.

The mutually perpendicular ordinary and extraordinary 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 speeds, which are defined by the dielectric properties along theto beyfastaxes of the inferior birefringent quartz crystalline wedge. The ordinary wave passes through the prism along the fast axis (which has a smaller index of refraction), while the extraordinary ray travels along the slower axis, which has a larger index of refraction. In quartz, the refractive index difference between the fast and slow axes is about 0.6%, and the fast axis is oriented perpendicular to the crystallographic axis of the wedge. Therefore, the ordinary wave passes through a section of quartz wedge perpendicular to the optical axis, while the extraordinary wave is oriented parallel to this axis.

An angular division orcutof the wavefronts occurs at the refractive index junction between the cemented quartz wedges, and the waves are spatially separated by an angle defined as thecutting angle. At this limit, ordinary and extraordinary waves also exchange identities (Figure 4). A wavefront (the common one) propagates from a medium of low refractive index to a second medium (the upper wedge) of higher refractive index and bends towards the normal (perpendicular to the optical axis of the wedge) according to Snell's law. The second wavefront (the extraordinary) leaves a medium with a high refractive index and enters a second medium with a lower refractive index, deviating the wavefront from the normal, but with the same angle as the first wavefront.

The cut angle and separation distance are constant for all wavefronts incident on the prism face, regardless of the entry point. The cutting direction of the wavefront is defined by the prism.cutting axis, which lies in the plane of the Wollaston prism and is parallel to the optical (crystalographic) axis of the lower section of the quartz wedge (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 slice axis, while the other will be oriented perpendicular to that axis. The cut angle is determined by the design of the prism components (quartz wedge angles, which are less than one degree of arc) and cannot be adjusted under the microscope. However, the cutoff distance is so small (generally less than a micrometer) that no observable beam splitting occurs in the light emerging from the prism.

During their journey through the lower quartz wedge in a Wollaston prism, ordinary and extraordinary wavefronts experience different refractive indices, as discussed above. As a result, the ordinary wavefront propagates through the crystal faster than the extraordinary wavefront. When the wavefronts exchange identities at the interface between the upper and lower quartz wedge, the ordinary wavefront becomes the extraordinary wavefront and vice versa. Furthermore, the wavefronts experience a phase shift in the lower half of the prism (due to differences in refractive index) that is exactly compensated for in the upper half when the geometric paths through the lower and upper half of the Wollaston prism are identical ( Figure 4). (B)). The wavefronts that pass through the prism moving away from the center experience a wider journey through the lower wedge of the prism before being cut (Figure 4(c)), or the upper wedge after being cut (Figure 4(a) ), before you leave. The extended distance traveled across a single prism wedge by the wavefronts eventually allows one of the waves (either normal (Figure 4(a)) or extraordinary (Figure 4(c)) to reach the quartz-air interface sooner. from the other). Along the face of the prism, there is a constant phase shift per unit length in the cutting direction that is equal, but opposite, for the ordinary and extraordinary wavefronts (Figure 4(a) and 4(c). )) . of the prism, the extraordinary wavefront emerges before the ordinary wavefront, while at the corresponding position at the other end, the ordinary wavefront emerges from the prism before the extraordinary wavefront.

If the wavefront incident on a Wollaston prism is polarized in an orientation parallel to the cut axis, no orthogonal splitting of the wavefront will occur and plane-polarized light will emerge from the prism. Likewise, if the incident polarized wavefront is oriented perpendicular to the prism's cutting axis, it will also emerge from the prism unchanged in 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 prism cutting axis. The electric vector of linearly polarized light entering from this angle is divided into two component vectors, each of which vibrates in the plane of the fast or slow axis of the quartz crystal and has the mean square amplitude (70.7%) of the front original waveform. . Both Wollaston and Nomarski prisms exhibit orientation-dependent properties. A beam of linearly polarized collimated light entering the opposite side of the prism (this time from the top) at a 45 degree angle will also produce ordinary and extraordinary orthogonal wavefronts. However, the polarization of the waves will be reversed.

When a Wollaston or Nomarski prism is placed between two crossed polarizers and examined with light transmitted through both the polarizers and the prism, a pattern of parallel interference fringes with a predominant central black band (fringe) can be observed (illustrated in Fig. Figure 5) . These patterns result from interference between the obliquely inclined ordinary and extraordinary wavefronts that emerge through the prism face. To the left and right of the central dark interference fringe, the peripheral fringes show increasing orders of the classical polarized interference color spectrum. Prismatic wedges designed for objectives with different focal lengths and numerical apertures are cut at shallower and shallower angles (as magnification and numerical aperture increase) to produce narrower bands of interference fringes. This concept is illustrated on the right side of Figure 5 for a series of fixed Nomarski prisms designed for successively larger objective magnifications (indicated in the figure).

If a first-order compensator (red plate) is added to the crossed polarizer sandwich in the diagonal position (not illustrated in Figure 5), the black fringe is replaced by interference colors showing subtraction (yellow) on one side and sum (blue) on the other side of the dark fringe's original position. Adding a second Wollaston or Nomarski prism over the first will offset the phase shifts (and resulting interference fringes) of the first prism over its entire length, causing extinction (illustrated in Figure 5; note that this effect can only be observed if the two prisms used for the experiment have the same cut angle). By translating one of the prisms laterally in relation to the other,inclination, or change in path length, will be introduced and can be observed across 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 smooth interference colors (starting with first-order yellow).

Interference fringes observed in a Nomarski prism 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 be inside the prism. The location of interference fringes for a Nomarski and Wollaston prism is calledinterference plane. Because the interference plane in a conventional Wollaston prism is located in the center of the prism, approximately on 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 prism's interference plane must coincide with and overlap the rear focal plane (also called the rear focal plane).diffraction plane) of the objective, which is usually located below the threaded mount inside a glass lens element.

Most manufacturers get around the lens aperture problem by employing Nomarski (occasionally referred to asModified Wollaston) for beam-slicing and recombination service in the condenser and objective focal planes, respectively. Due to a specialized design, as discussed below, Nomarski prisms have an interference plane that is offset several millimeters outside the prism, rather than encompassing the wedge elements as in the traditional Wollaston design. Nomarski prisms need not be physically located in the focal plane of the objective or condenser, but can be placed some distance away. Although Nomarski prisms do not produce better contrast, they avoid the potential problem of interference fringes becoming visible in the field of view. It should be noted that Leica Microsystems once produced a popular microscope known as theblacksmith tdifferential 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 prism surface. However, the second wedge is modified by cutting the quartz crystal so that the optical axis is oriented obliquely to the flat surface of the prism. When the wedges are combined to form a birefringent composite prism, the focal plane (and the interference fringes that are produced when polarized light passes through the prism) lie outside the prism plate, as described above and illustrated in Figure 6. This effect This is because shear now occurs at the air-quartz interface of the lower wedge, and refraction at the interface between the quartz wedges causes the shear wavefronts to converge to a crossing point outside the prism. The actual position of the focal plane of the Nomarski prism can be adjusted by several millimeters by changing the oblique angle of the optical axis on the second quartz wedge used to construct the prism.

Although Nomarski prisms are widely used as objective prisms in modern differential interference contrast microscopes, there are fewer spatial restrictions for condenser prisms, which can often be precisely positioned within the plane of aperture. Therefore, sometimes a conventional Wollaston prism can be inserted into the microscope condenser, but in many cases a Nomarski prism is used. When a Nomarski prism is used in the condenser, the prism is designed to produce an interference plane located much closer to the prism than those constructed for use with objectives. As a result, in addition to being mounted in frames with 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 known assecondary,assistant, ocompensatingcomposite prism) acts as a primary beam splitter to cut the polarized wavefront, while the objective prism (thedirectorprism) recombines the separate waves and regulates the degree of delay between the ordinary and extraordinary wavefronts.

It is important to remember that aligning the microscope for Köhler illumination is a critical and necessary step to ensure correct positioning of the interference planes of the Nomarski prism to match the conjugate aperture planes of the condenser and objective. the center, orzero orderThe interference fringe, 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 microscope alignment.

DIC wavefront relationships and images

After leaving the condenser Wollaston or Nomarski prism in the aperture plane, the clipped ordinary and extraordinary coherent wavefronts are focused by the condenser lens elements and travel through the specimen before being captured by the objective. Along their paths between the condenser and the target, the wavefronts remain parallel to each other and are separated by acutting distancederived from the geometric constraints of the condenser prism. The spatial separation between the wavefronts (cutoff distance) varies with the numerical aperture of the condenser and objective, but has practical limits between 0.1 and 1.5 micrometers, a linear range designed to be slightly less than (or in some cases equal to) the lateral resolution of the target. Resolution in differential interference contrast can be increased (at the expense of contrast) by reducing the cutoff distance to about half of the target's maximum resolution.

Most microscope manufacturers compromise the tradeoff between cutoff distance and resolution (and contrast) and produce prisms that have a maximum cutoff distance of about 0.6 micrometers for lower magnification (10x) objectives down to a minimum of 0. 15 micrometers for higher magnification objectives. (60x and 100x). However, regardless of the shear distance, it is important to note that closely spaced pairs of wavefronts, spatially distributed across the microscope aperture, sample every point in the sample to ultimately provide a dual-beam interference in the plane of the microscope. image.

When undisturbed by the presence of a sample, pairs of coherent wavefronts experience identical light path differences between the sample and image planes and arrive at the objective rear focal plane with 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 that has an electric vector vibration orientation identical to that of the transmission axis of the substage polarizer. The linearly polarized wavefronts leaving the objective prism are blocked by the second polarizer (or analyzer), which has a transmission axis oriented perpendicular to that of the polarizer (Figures 7(a) and 7(b)). As a result, the background of the image seen in the visual field appears too dark or black, a condition calledextinction.

With no specimen-induced phase shifts, the beam splitting action of the condenser prism is exactly matched and reversed by the beam recombination effect of the Nomarski objective prism to finally produce linearly polarized light. In other words, when the microscope is set up correctly for Köhler illumination (a critical prerequisite for high-resolution differential interference contrast microscopy), the condenser and objective work together to project an image of the light source and the prism of the condenser on the objective prism. The Nomarski objective prism, whose orientation is inverted with respect to the condenser prism, introduces a phase shift that exactly compensates for the linear phase shift between the wavefronts produced by the condenser prism. This action occurs for all paired wavefronts across the microscope aperture. The axes of the condenser and the objective prisms are parallel to each other and oriented at an angle of 45 degrees to the transmission axes of the crossed polarizers (polarizer and analyzer). The axis of orientation of the two prisms is calledcutting axis, an important concept that defines the axis of lateral separation between the ordinary and extraordinary wavefronts from the moment they leave the condenser prism until they are recombined by the objective prism and reach the image plane.

If the paired coherent wavefronts encounter a phase gradient present in the sample as they pass from the condenser to the target, wavefront distortion is induced and the waves will undergo a phase shift along the clipping axis. optical paths (although they do not change). in the polarization occurs). Upon reaching the target prism, the phase-shifted paired wavefronts recombine to generate elliptically polarized light (in contrast to the linearly polarized light that occurs in the absence of a sample). The resulting electrical wavefront vector, which is no longer flat, traces an elliptical path as it traverses the region between the target prism and the analyzer (as illustrated in Figure 7(c)). Because a component of the elliptical wavefront is now parallel to the analyzer's transmission axis, a portion of the wave will pass through the analyzer and produce plane-polarized light that will have finite amplitude and may ultimately generate intensity in the image plane.

In summary, the optical path gradients in the sample induce phase shifts in the coherent paired wavefronts cut by the condenser prism and passing through parallel paths. 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, across the entire sample field, the presence or absence of phase gradients creates a combination of linearly and elliptically polarized wavefronts that selectively pass through the analyzer according to the azimuths of their planes of vibration. The wavefronts that can pass through the analyzer are all parallel planes and can image the sample amplitude through interference in the image plane. When the objective prism exactly compensates for the effects of the condenser prism (as in Köhler illumination), the analyzer blocks wavefronts originating from all spatial locations in the field without phase shifts (no phase gradients) of the sample). The resulting background seen in the field of view is dark (displays full extinction), with the exception of regions that show a steep sample refractive index or thickness gradients, which appear much lighter (usually in outline form). The perceived image looks very similar to the images generated by the classic and simple darkfield lighting technique.

These concepts are graphically represented in Figure 8, which presents the phase relationships between the clipped wavefronts after passing through a phase sample (which has a higher refractive index than the surrounding medium) and its amplitude profile (or intensity ) corresponding in the image plane. Mutually perpendicular wavefronts (labeledS(1)yS(2); see Figure 8(a)) passing through the sample are distorted and show localized regions of phase delay (calleddifferential phase delays). The Nomarski objective prism recombines the wavefronts 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 ordinary and extraordinary components) reconstructed in the image plane along the slice axis are illustrated in Figure 8(a) when the dual prism instrument setting was set to maximum extinction. phase delay (Fi) introduced into the wavefronts by the sample is indicated on the ordinate (measured in nanometers), while the dip width along the slice axis (x) represents the diameter of the enlarged sample (in this case, a single drop of oil).

After the phase-delayed wavefronts of Figure 8(a) have been transmitted through the analyzer and brought together by constructive and destructive interference in the image plane, the resulting intensity distribution can be plotted as an amplitude diagram along the axis cutting edge (Figure 8 (b)). For the symmetrical oil drop sample considered in this example, the plot of amplitude versus distance along the slice axis produces a dark central cavity flanked on each side by bright regions (see Figure 8(b)). A digital image of the actual sample viewed under the microscope (Figure 8(c)) reveals spherical microdomains that have bright edges superimposed on a black background and a dark interference fringe in the center. The images shown 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 that divide the hemispherical oil droplets will be oriented perpendicular to those illustrated in Figure 8(c).

Introduction of bias delay

On a differential interference contrast microscope set to maximum extinction, the field of view is rendered with a dark, almost black background and shows very high sensitivity for regions of the sample that have increasing and decreasing phase gradients. As illustrated in Figure 8(c), some specimen details are obscured or very difficult to resolve in this configuration, and the zero-order interference fringe can often be seen crossing highlighted features. In practice, the objective Nomarski prism is laterally displaced along the slice axis to uniformly alter the relative phase shift of the ordinary and extraordinary wavefronts passing through the sample. Therefore, the orientation of the polarization vector of light emerging from the objective prism can be adjusted from linear to various degrees of elliptical and even circular. The change of phase shift of the ordinary wavefront relative to the extraordinary wavefront through translation of the target prism is often calledintroduction of bias delayin DIC microscopy.

As the objective Nomarski prism is shifted laterally (to the left or right of the microscope's optical axis), the pairs of wavefronts contributing to the background become increasingly slower and out of phase with each other. As a result, the degree of elliptical polarization increases on wavefronts entering the analyzer and the background intensity progressively changes from black to lighter mid-grey tones. Furthermore, the introduction of the polarization delay changes the position of the zero-order interference fringe and produces corresponding changes in the intensity levels of the phase gradients in the sample. This results in orientation-dependent bright reflections and dark shadows being superimposed on the now lighter background (which has a color often calledzero order gray). The final DIC image does not depend on the optical path difference being introduced solely by the translation of the objective prism, and the same result can be obtained when the condenser prism is moved along the optical axis of the microscope. However, in most instruments it is much more convenient to produce bias delay by changing the position of the objective prism rather than prisms housed in a condenser tower.

Intensity gradients introduced into the specimen by changing the bias delay occur along the cut axis of the condenser and objective prisms and generally appear to originate from a 45 degree angle (northwest to southeast or vice versa) when the specimen is viewed. in the eyepieces (see Figure 8(f)). Note that the gradients illustrated in Figure 8(f) were recorded using an upright microscope. Inverted microscopes produce intensity gradients oriented perpendicularly to those seen with upright microscopes. Moving the prism in one direction or the other will affect the displacement of the interference fringe and vary the phase relationship (delayed or lead) between the ordinary and extraordinary wavefronts, thus reversing the orientation of the shadow cast on the sample.

The net result of introducing bias lag is to render the sample image in pseudo-three-dimensional relief, where regions with increasing optical path difference (inclined phase gradients) appear much brighter (or darker) and those that exhibit a decreasing path length appear in reverse. Sample features appear similar to raised plateaus or sunken valleys, depending on the orientation of the phase gradient, which is a hallmark of differential interference contrast. However, the three-dimensional appearance corresponds only to phase gradients and should not be confused with the actual sample geometry (as is often the case). In some situations, actual topographical features are also sites of phase gradient change, but in the absence of information obtained as a result of independent investigations, this fact should not be assumed.

The introduction of polarization delay in differential interference contrast microscopy is illustrated in Figure 8(d) to Figure 8(f) for a phase sample consisting of several hemispherical oil droplets. When the microscope is set to maximum extinction, the ordinary and extraordinary wavefronts show a phase shift along the slice axis, but do not show a phase difference in the background regions (Figure 8(a)). Adding the target prism translation bias delay changes the relative phase of one wavefront with respect to the other (Figure 8(d)), but the wavefront shear remains the same. After interference in the image plane, the resulting graph of amplitude (or intensity) as a function of the cutoff distance (Figure 8(e)) shows a bright edge region on one side of the oil droplet and a dark region on the other side. opposite side . When viewed under the microscope, the specimen exhibits a shadow-cast appearance, as if it is being illuminated from a highly oblique angle (see Figure 8(f)). To observe the difference between maximum extinction and bias delay addition, compare the sample images shown in Figure 8 (c) and (f). The shadow's orientation, which depends on the cutting axis (double-headed arrows in Figure 8(c) and (f)), can be reversed by translating the target prism the same amount in the opposite direction.

Traditionally, bias has been introduced into the differential interference contrast microscope by translating the Nomarski objective prism back and forth along the optical axis using a fine-adjustment knob located on the end of the mounting frame (usually located on the holder housing). microscope objectives or in an intermediate tube). An alternative technique, which is gaining in popularity, is to mount a quarter-wavelength delay plate in a fixed orientation between the polarizer and the condenser prism (calledof SenarmontDIC compensation). At maximum extinction, the fast axis of the delay plate aligns with the transmission axis of the polarizer and both optical units can be (and usually are) within the same housing at the base of the microscope. An alternative location for the Sénarmont compensator, on microscopes equipped with the appropriate intermediate tube, is between the objective prism and the analyzer.

To introduce polarization using the Sénarmont compensator, the polarizer drive shaft is rotated (up to plus or minus 45 degrees) with respect to the fast axis of the delay plate, which remains fixed at a 90 degree angle to the analyzer. drive shaft. When the fast axis of the compensator coincides (is parallel) with the transmission axis of the polarizer, only linearly polarized light passes through the Sénarmont compensator into the condenser prism. However, when the polarizer drive shaft is rotated, the wavefronts emerging from the quarter-wavelength delay plate become elliptically polarized. Rotating the polarizer in one direction will produce right elliptical polarized light, while rotating the polarizer in the other direction will change the path of the vector to generate a left elliptical sweep.

When the orientation of the polarizer's transmission axis reaches plus or minus 45 degrees (equivalent to a quarter wavelength delay), the light passing through the compensator becomes circularly polarized (again left or right). . Since elliptically or circularly polarized light represents a phase difference between the ordinary and extraordinary wavefronts emerging from the Sénarmont compensator, polarization is introduced into the system when the wavefronts enter the condenser prism and intersect. Positive bias is achieved by rotating the polarizer in one direction, while negative bias is introduced by rotating the polarizer in the opposite direction.

Regardless of whether polarization is introduced in a differential interference contrast system, translating the Nomarski objective prism, or rotating the polarizer in a Sénarmont compensator, the net result is the same. As discussed above, in a properly configured and aligned microscope for Köhler illumination, the optical system (condenser and objective) transfers an image from the light source and condenser prism to the second inverted Nomarski prism located in the focal plane. the target. The linear phase shift on the condenser prism face is precisely compensated for by an opposite phase shift on the objective prism. Translating the objective prism along the slice axis does not change the phase shift distribution, but adds or subtracts a constant phase difference across the microscope aperture. Likewise, polarizer rotation in a Sénarmont compensator also introduces a variable and controlled phase difference. The combined prism system allows imaging to occur with the same polarization delay for each pair of wavefronts projected from the condenser aperture, regardless of the path taken by the sample to reach the target.

A series of digital images recorded in DIC using a one-twentieth to one-quarter wavelength polarization delay range at several intermediate steps is shown in Figure 9. The sample is a 15-micron section of fixed, mounted murine intestine containing regions of fluctuating thickness. Rendering of sample details and pseudo-3D shading effects are more pronounced at lower bias delay values ​​(Figure 9(a) and 9(b)), but fine contrast sample detail and image definition deteriorate with the bias delay. increases (Figure 9(c) to 9(f)). At the highest polarization delay value (quarter wavelength; Figure 9(f)), contrast is extremely poor and little structural detail is visible. For this particular specimen, the ideal delay range appears to be between one-twentieth and one-twelfth of a wavelength.

As the optical path gradient in a sample increases, the contrast of the image also increases. Changing the bias lag by varying degrees can also produce significant contrast fluctuations in the sample, as seen 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 rotation of the polarizer in a Sénarmont compensator, is on the order of less than a tenth of a wavelength. However, this value is highly dependent on sample thickness, and the useful range of polarization delay for biological samples is between one-thirtieth and one-quarter of a wavelength. Contrast in samples that have very large optical gradients can often benefit from even larger polarization delay values ​​(up to full wavelength). The introduction of polarization delay in a differential interference contrast microscope allows phase samples to be observed more easily and greatly facilitates imaging efforts with traditional film or digital camera systems.

DIC microscopy delay compensation plates

The polarization delay between ordinary and extraordinary wavefronts in differential interference contrast can also be manipulated through the use of compensators originally conceived as quantitative delay measuring devices and contrast enhancement elements for polarized light microscopy. Offset plates offer more control to adjust the contrast of sample details against background intensity and color values, and allow finer adjustment of the polarization value between wavefronts. These birefringent components are also often used foroptical coloringof transparent specimens, which are normally represented in a limited range of grayscale values.

When a standard objective Nomarski prism is translated along the optical axis of the microscope beyond quarter-wavelength path differences, both the sample and background features assume a spectrum of Newtonian interference colors similar to those observed in light microscopy. polarized light. The specimen and background are optically stained with a color transition that migrates through a range of values ​​from gray through white, yellow, red-blue and higher orders. Optical coloring produces beautifully colored and dramatic images, but is of limited use for scientific applications. Typically, optimal sample contrast is limited to the delay range of one-twentieth to one-quarter of a wavelength.

Compensators can be inserted in the optical path of a DIC microscope between the objective prism and analyzer or the polarizer and condenser prism. Many microscopes have a slot located in the middle tube or lower stage condenser housing designed for this purpose. Addition of a first-order compensator (often calledfull waveofirst order redplate) with a delay value equal to a full wavelength in the green region of visible light (approximately 550 nanometers), introduces an interference color spectrum into the sample and background. With the compensator installed, the green light cannot pass through the analyzer because it leaves the delay plate linearly polarized with an electric field vector that has the same orientation as the polarizer. However, the wavefronts in the red and blue spectral regions are delayed by less than one wavelength and become elliptically polarized, allowing a component to pass through the analyzer. As a result, these colors blend together to form a magenta background in the field of view.

Therefore, when viewing a sample in white light with differential interference contrast optics and a first-order compensator, the background appears magenta while the image contrast appears second-order blue and first-order yellow. sample order (depending on orientation). Newtonian interference color spectrum. With the compensator installed, small variations in the polarization delay obtained by translation of the Nomarski prism (or rotation of the polarizer in a Sénarmont compensator) produce rapid changes in the interference colors observed in structures with large gradients. This technique is useful for introducing color (optical staining) into regions with high refractive index boundaries, such as cell membranes, large intracellular particles, cilia, and the nucleus. Interference colors that show specimen characteristics can be compared to values ​​in a Michel-Levy color table 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 three-dimensional pseudo-relief using optical DIC techniques. Figure 10(a) shows projections on the edge of a ctenoid fish scale, while the mouth of a canine hookworm (canine hookworm) appears in Figure 10(b). Colorful scales on the wings of the large leopard moth (Ecpantheria scribonia) are shown in Figure 10(c). In all cases, the Nomarski prism was translated through the optical axis of the microscope by a polarization delay value greater than a full wavelength. While these images do not reveal hidden scientific information related to the specimens, they have the potential to promote the DIC light microscopy technique as a legitimate bridge between science and art.

On microscopes equipped with a Sénarmont compensator to introduce polarization into a differential interference contrast optical system, a full-wave delay plate can be added to optically color the sample with Newtonian interference colors and provide more quantitative information about path differences. . As discussed above, the Sénarmont compensator is often employed in DIC microscopy to obtain precisely measured polarization delay levels, but the device is also useful for monitoring the alignment of optical components. In video-enhanced DIC microscopy, Sénarmont compensators are often used to optimize contrast in sample details below the resolution limit of the microscope.

DIC image interpretation

Among the most notable and recognized aspects of the images obtained with differential interference contrast microscopy is the high degree of visible relief, manifested by a shadow projection effect that confers a pseudo-three-dimensional realism. In general, samples appear as if illuminated from a low angle with a highly oblique light source, reminiscent of results obtained with traditional oblique illumination or Hoffman modulation contrast. However, with caution, images should always be interpreted with the understanding that shadows and reflections in shadow projection representations only indicate the sign and orientation of the phase gradients slope or optical path and do not necessarily reveal geometric parameters. or precise topography.

Looking at the orientation of the shadow present in almost all images produced through bias delay, the direction of optical shear is obvious and can be precisely defined as the axis that connects the regions showing the highest and lowest intensity values. Another consideration that must be taken into account is the relationship between the specimen and its surroundings, because shadow directions are often reversed for details in the specimen that have 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 often have the appearance of high elevations (peaks), while inclusions of lower refractive index (e.g., refractive index vesicles) . pinocytosis, aqueous vacuoles, and lipid droplets) appear to be deep depressions (craters).

The level of contrast given to the sample phase gradients (and the degree of pseudo-three-dimensionality) by the differential interference contrast is a function of the amount of polarization delay introduced into the optical system by the translation of the Nomarski prism or its rotation. the polarizer in a de Sénarmont. compensator. As the cut axis is fixed by the design of the Nomarski and Wollaston prism and other constraints involved in the orientation of the wavefront for differential interference contrast, the direction of the axis cannot be changed to affect sample contrast through a simple setting. under the microscope. However, the relative phase delay between ordinary and extraordinary wavefronts can be reversed by relocating the objective prism from one side of the microscope's optical axis to the other (changing the bias delay from negative to positive or vice versa). This operation can also be performed by turning the polarizer to the corresponding negative value on a Sénarmont compensator. When the phase delay is changed as described, the orientation of the light and dark edges of the sample is reversed by 180 degrees. In essence, the only mechanism available to the microscopist to change the cutting axis relative to the specimen is to reorient the specimen itself, a maneuver that benefits from the use of 360-degree rotating circular stages (primarily designed for microscopy). polarized light).

In differential interference contrast images, the intensity of shadows and highlights is greater along the microscope's cross-sectional axis, and regions of constant refractive index have identical intensity values ​​to those of the background. When a sample with spherical geometry (Figure 8(f)) is examined from various azimuths for contrast on the edges that limit the background, the contrast is minimal in regions perpendicular to the slice axis. In fact, the differences in contrast between the sample and the background gradually decrease until they reach a minimum value in the direction that defines the axis of the zero-order interference fringe (perpendicular to the cut axis). In Figures 8(c) and 8(f), the regions of the sample that show the lowest level of contrast (indicated by an unmarked white arrow) are the edges in the central area of ​​the oil drops found exactly perpendicular to the cut axis. To verify this concept, compare an edge where the interference fringe meets the background in the image recorded at maximum extinction (Figure 8(c)) with the corresponding region in the image generated after introducing the polarization delay (Figure 8(f)). . In the areas marked by the unlabeled white arrow, the ordinary and extraordinary wavefront distortions have aligned profiles, which are subtracted in the analyzer to cancel the residual delay and produce an intensity value that exactly matches the background.

Each specimen examined under differential interference contrast microscopy will have an optimal polarization delay setting that generates a maximum level of contrast in the final image. Very thin samples that exhibit a shallow refractive index gradient, such as live cells in culture, generally benefit from similarly low bias settings, only slightly greater than the largest phase shift present in the sample (on the order of about one-twentieth of a wavelength, or about 30 nanometers). However, thicker samples generally require higher polarization settings (up to a quarter wavelength) at large condenser apertures to produce satisfactory results, often via optical clipping. Because many specimens are composed of features that show a variety of different sizes and refractive indices, the optimal polarization delay setting is often a compromise.

The azimuthal effects of orientation phenomena on differential interference contrast for various specimens are shown in Figure 11. In all cases, the slice axis is directed northwest to southeast, but is not indicated on the individual digital images. Figures 11(a) and 11(b) illustrate the periodic pore spacing and striations exhibited by an assembled diatom frustule.gyrosigma attenuation. When the long axis of the frustule is oriented perpendicular to the cut axis (Figure 11(a)), the pores appear to merge into a series of closely spaced grooves and do not resolve individually. In contrast, reorientation of the frustule in a direction parallel to the cut axis reveals the geometry of the button-shaped pore structure in this species.

The region of the thorax containing freshwater gill ribsdaphnia(water flea) is shown in Figure 11(c). In this orientation, the individual rib structures are clearly visible (perpendicular to the cutting axis) and are connected to a common spine, which appears as a long series of protuberances parallel to the cutting axis. When the specimen is rotated 90 degrees (Figure 11(d)), much of the rib structure loses contrast and the spine bumps merge into a single ridge. Finally, inclusions in a ctenoid fish scale lack contrast and are difficult to distinguish when superimposed on the striated spines of the body (Figure 11(e)). Rotating the sample to bring the spines parallel to the cutting axis (Figure 11(f)) reduces their contrast and makes the inclusions clearly visible.

Several factors must be considered when adjusting the polarization delay to produce optimal sample contrast. The bias level that must be entered to make a sample slope or edge as dark as possible also produces the maximum possible contrast between the sample and the background. Therefore, for each specimen, a particular objective prism (or Sénarmont compensator) configuration will generally introduce the greatest degree of contrast. When this value is exceeded, the contrast decreases. Thicker specimens, which often suffer from light scattering artifacts, often require a larger polarization delay setting (up to a full wavelength) than thinner specimens to achieve extinction in regions that have significant phase gradients. Finally, when the aperture size of the iris diaphragm in the condenser exceeds 75% of the rear aperture of the lens, contrast is reduced as a result of excessive scattering of light in the optical system. With this difficulty in mind, be careful when opening the condenser aperture to perform optical sectioning experiments.

optical sectioning

The ability to image a sample in differential interference contrast with a large condenser and objective numerical apertures allows the creation of remarkably shallow optical sections from a focused image. Undisturbed by halos and distracting intensity fluctuations from bright regions in the lateral planes far from the focal point, the technique produces sharp images that are carefully cropped from a complex three-dimensional phase sample. This property is often used to obtain sharp optical sections of cell contours 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 an important role in defining image contrast and resolution. Reducing the aperture size increases the depth of field and overall image sharpness while improving contrast. However, if the diaphragm is too closed, diffraction artifacts become apparent and resolution is sacrificed. Often, the optimal aperture setting is a compromise between accurately representing specimen detail with sufficient contrast and maintaining the resolution necessary to image tiny features and avoid diffraction artifacts.

The high-performance differential interference contrast microscope's optics produce excellent contrast with the partially closed condenser iris diaphragm (approximately 70 percent the size of the objective's rear aperture), but also perform excellently when the diaphragm is closed. the lens aperture. To achieve the optimal balance of resolution and contrast for optical sectioning, the microscope must be correctly set up for Köhler illumination and the prism components and polarizers must be precisely aligned. High numerical aperture objectives designed for oil immersion should only be used to image specimen slides that have oiled bottoms in the condenser.

At high numerical magnifications and apertures (e.g., 100x and 1.4), the depth of field in differential interference contrast approaches a cutoff value of about 400 nanometers (0.40 micrometers; about 1.5 times the cut). target resolution). 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 settings used to obtain the images in Figure 12 was approximately 0.5 micrometers. The bacteria are clearly visible on the top surface of the cell (Figure 12(a)), as are the parallel, spiral bumps on the membrane that closely resemble the texture of human fingerprints. In the lower central part of the figure there is also a bulge in the membrane corresponding to the nucleus. As the focal plane is moved into the cell (Figure 12(b)), details of the nuclear structure and intracellular particles become visible. Finally, at the lower boundary where the cell membrane rests on the surface of the microscope slide (Figure 12(c)), numerous folded ridges (similar to those seen on the upper surface) are revealed.

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

A significant amount of current research and theory development in differential interference contrast is focused on the technique's impressive optical cutoff characteristics. Ultimately, a quantitative estimate of the three-dimensional refractive index profile of a sample can be obtained using computer models. In addition, current research is also directed towards developing new models for DIC optical components and partially coherent transmitted light imaging.

conclusions

Differential interference contrast microscopy is basically a beam shear interference system in which the reference beam is clipped by a minuscule amount, typically slightly less than the diameter of an Airy disc. In fact, each sample point is represented by two overlapping Airy disks in the final image, one lighter and one darker than the background. The basic microscope system, first developed by Francis Smith in 1955, is a modified polarized light microscope with two Wollaston prisms added, one in the front focal plane of the condenser and the second in the rear focal plane of the objective.

Later modifications, suggested by Georges Nomarski, allowed the prisms to be physically located away from the conjugate planes of the optical aperture. The condenser prism converts each wavefront illuminating the sample into two slightly offset parallel beams that are polarized orthogonally to each other, while the objective prism serves to recombine the beams. The combination of these two prisms, which the optical system reflects off each other, is a key feature in Differential Interference Contrast's ability to form sharp images with 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 sample, resulting in an elliptical polarization for the recombined beam leaving the Wollaston target prism. Polarization delay can be introduced into the system by translating the objective prism along the optical axis of the microscope or by combining a quarter-wavelength plate with the polarizer or analyzer. Thus, optimum contrast, field brightness and sensitivity can be achieved simply by turning a control knob.

The resulting DIC images have a shadow cast appearance that effectively shows the gradient of the lightpaths for low and high spatial frequencies. Sample regions where the lightpaths increase along a reference direction appear brighter (or darker), while regions where the path differences decrease appear in reverse contrast. Steeper gradients in the optical path difference result in greater contrast. A wide variety of samples 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 major imaging advantages of differential interference contrast microscopy is that, unlike darkfield or phase contrast, the image of the smallest features in the specimen is not obscured by adjacent regions that have large optical gradients. In addition, the image's drop shadow appearance on a neutral gray background, along with the sensitivity to images of very small features next to much larger ones (e.g., tiny appendages of living cells or dynamic inclusions and moving organelles within 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 contributed to the technique's great popularity.

Contrast in DIC microscopy is directional, exhibiting a maximum along the slice axis and a minimum in the orthogonal direction. The separation direction of the Airy disc (with an average peak-to-peak separation of one-half to two-thirds of the disc radius) coincides with the microscope cut axis, which is the direction of maximum contrast. As a result, the DIC contrast transfer function (CTF) is also directional along the cutting axis. The lateral shift between two wavefronts passing through the sample is approximately half of the resolution limit of the target. This small degree of shear, induced by the Wollaston or Nomarski prism, has an action similar to that of a high-pass filter for contrasting spatial frequency details in the sample. The corresponding modulation transfer function (MTF) closely follows that observed in brightfield illumination for high spatial frequencies, but shows a strong decline for sample features exceeding several micrometers in size (lower spatial frequencies).

Samples suitable for observation in DIC include fluid smears, live 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 gained by examining mixed phase amplitude and amplitude samples such as naturally pigmented protists, algae, and lightly stained histological samples. The technique is often used in combination with fluorescence microscopy to reveal cell morphology associated with fluorescent regions. When combined with enhanced video techniques (callsVEC-DICfor video-enhanced differential interference contrast), DIC can be used to produce images of structures with dimensions below the optical resolution of the microscope.