The proper adjustment and alignment of differential interference contrast (DIC) optical components is critical to imaging performance, so it is imperative that the microscopist recognize misalignments and component mismatches, and take the necessary steps to correct these errors. This interactive tutorial examines conoscopic and orthoscopic viewfields in a DIC microscope under a variety of configurational motifs, and discusses many of the important aspects recommended for satisfactory microscope alignment.
The tutorial initializes with the virtual DIC microscope placed in Conoscopic viewing mode (simulating a virtual phase telescope or Bertrand lens), and only the polarizer and analyzer (transmission axes crossed) inserted into the optical pathway. In addition, the condenser diaphragm has been closed to 80 percent of it widest aperture size upon initialization. To operate the tutorial, first select a DIC technique, either Nomarski or de Sénarmont, from the set of Imaging Mode radio buttons positioned to the left of the viewport. Next, select an objective from the Objective Magnification pull-down menu (the default is 10x). Note that when a new objective is chosen, the size of the condenser aperture diaphragm observed in the microscope viewport increases or decreases proportionately. Use the Condenser Diaphragm slider to alter the opening size of the aperture diaphragm.
To install a Nomarski prism in the optical train, use the Objective Prism or Condenser Prism radio buttons. When one of these radio buttons is selected, the viewport displays the conoscopic image of a single Nomarski prism, which varies in size depending upon the objective selection. If the Objective Prism has been selected, the slider changes to display the text: Objective Prism Translation, when the microscope is in Nomarski mode, orPolarizer Orientation when the microscope is in de Sénarmont mode. If the Condenser Prism has been selected, the Polarizer Orientation text will appear when the microscope is in de Sénarmont mode, but the slider will be inactivated when the microscope is inNomarski mode.
In either Nomarski or de Sénarmont imaging modes, the slider can be utilized to alter the bias retardation that is introduced into the optical train. In Nomarski mode (only when theObjective Prism is selected), the Objective Prism Translation slider is initially in the central position with no bias added. Translating the slider to the right introduces Negative Bias, while moving the slider to the left of center produces Positive Bias. The tutorial operates in an identical manner in de Sénarmont mode, except that either prism can be selected, and the Polarizer Orientation slider also indicates the number of degrees that the polarizer is rotated from the central position.
To add both prisms (condenser and objective) to the virtual DIC microscope optical train, select the Both Prisms radio button in either Nomarski or de Sénarmont imaging mode. The bias can then be adjusted using either the Objective Prism Translation (Nomarski mode) or Polarizer Orientation (de Sénarmont mode) slider. In order to add a first-order compensation plate to the optical pathway, place a check mark in the Compensatorcheckbox. In all cases discussed above, the virtual DIC microscope can be placed in orthoscopic mode by selecting the Orthoscopic radio button. The viewport will then reveal how the field appears when the virtual Bertrand lens is removed from the optical pathway.
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 prisms, 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. 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 1(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 1(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 or a prism confined to a fixed mount (for systems utilizing de Sénarmont bias retardation). 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 1(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 1(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 white10 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 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 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 2(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 2(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 2(a) with Figure 2(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 1(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 1(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 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 1) 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 dark 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 1(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 3(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 3(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 3(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 3(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.
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.