Specimens that are nearly transparent and colorless may be almost invisible when viewed in the stereomicroscope using traditional transmitted (diascopic) brightfield illumination techniques. This occurs because light diffracted by minute specimen detail is a quarter-wavelength out of phase with direct light passing through the specimen when both are recombined in the intermediate image plane, a classical phenomenon that seriously reduces contrast in brightfield images.
However, if the illumination is directed so that it originates from a single azimuth and strikes the specimen from an oblique angle, details in the specimen may be revealed with much greater contrast and visual clarity than when the light is allowed to pass directly through specimen features along the optical axis of the microscope. Phase and refractive index gradients in the specimen deflect the light rays by diffraction, reflection, and refraction, so that only the zeroth order (undiffracted) and one or two sidebands of diffracted light can recombine at the image plane. This produces a relief-like specimen pattern having regions displaying shadows and highlights, much like that observed with the differential interference contrast (DIC) technique in compound microscopes.
Presented in Figure 1 is a modern stereomicroscope illumination stand (the Nikon Oblique Coherent Contrast (OCC) diascopic model), which is designed to illuminate specimens through a transitional mechanism ranging from axial brightfield to highly oblique off-axis light rays that render transparent specimens visible in a scheme closely resembling darkfield. The stand contains both a high and low numerical aperture condenser enabling utilization of the entire stereomicroscope objective magnification (generally 0.5x to 2x) and numerical aperture (0.07 to 0.21) range. Oblique illumination is achieved by means of a sliding diaphragm that shields the center of the light beam to produce a partially coherent light source, which is projected obliquely onto the specimen, producing a high contrast image. The diaphragm position is controlled by means of a rotating knob that can be employed to adjust the obliquity of illumination.
Specimens are placed on the glass stage of the Nikon OCC diascopic stand and can be illuminated either in brightfield, darkfield, or varying degrees of oblique illumination, by rotating the diaphragm control knob. A series of digital images captured at successive levels of oblique illumination are illustrated in Figure 2 for a semi-transparent Nemathelminth (hookworm; Ancylostoma caninum) specimen. As is evident in Figure 2(a), the unstained hookworm is semi-transparent, and very little detail is revealed under brightfield axial illumination. However, when the sliding diaphragm is rotated into the light path, increasing degrees of oblique illumination can be achieved (Figures 2(b), 2(c), and 2(d)), with the most extreme position corresponding closely to darkfield illumination from a single azimuth. The change in specimen contrast possible with this illumination stand is most dramatically portrayed by comparing Figures 2(a) and 2(c), which were produced in brightfield and highly oblique coherent contrast, respectively.
One of the major design criteria of the Nikon oblique stereomicroscope illumination stand is to enable enhancement of image contrast, such as might be achieved with iris diaphragms, while maintaining a high condenser numerical aperture capable of matching that displayed by apochromatic objectives. Many stereomicroscopes having the parallel or common main objective (CMO) design are coupled to illumination stands that are equipped with iris diaphragms, either built-in or available as accessories. In order to increase depth of field, the diaphragm aperture is often reduced in size, which also serves to increase image contrast in diascopic illumination. Unfortunately, reducing the iris diaphragm size also renders the image-forming light rays more coherent, and compromises the resolving power of the microscope by reducing the working numerical aperture of the objective.
The oblique coherent contrast illumination system combines the effect of oblique illumination with the coherence enhancement obtained by reducing condenser aperture diaphragm size, and creates images in the stereomicroscope that have an appearance similar to differential interference contrast images produced by a compound microscope. Instead of using an iris aperture diaphragm, the oblique coherent contrast system employs a baffle that acts as a sliding diaphragm. The linear diaphragm mechanism behaves as an iris would if placed at the same position, except that the objective retains most of its resolving power. This can be achieved because the numerical aperture of the illumination cone is reduced, but that of the objective lens is not.
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The illumination pathways that characterize the Nikon oblique stereomicroscope system are illustrated in Figure 3, which also presents the light pathway for normal brightfield (diascopic) conditions. A collector lens system focuses an image of the lamp filament onto a mirror positioned at a 45-degree angle with respect to both the lamp and specimen optical axes. The sliding diaphragm is a baffle that can be translated across the surface of the mirror to block light that would ordinarily pass directly through the specimen, ensuring that only oblique light is utilized to illuminate specimen detail. A condenser lens positioned above the mirror maintains the coherence of illumination by bringing the sharp edge of the baffle into focus at the rear focal plane of the objective. The rear pupil of the objective will be only partially filled with light, as determined by the sliding baffle position (functioning as an iris diaphragm).
With this system, the full numerical aperture of the objective is maintained. One of the advantages of the Nikon oblique system (over other oblique illumination techniques) is the ease of translating between brightfield, oblique, and darkfield illumination by means of a single adjustment knob, which controls the position of the sliding baffle. The appearance of the specimen with the baffle moved away from the mirror is equivalent to that seen in normal brightfield illumination. With the baffle moved up to the top of the mirror, the image will be similar to darkfield, and in between these extremes, true oblique illumination conditions are obtained. The degree of obliquity is easily adjusted to suit the specimen and the type of detail that is desired.
Oblique illumination is similar in many aspects to the darkfield technique except that, instead of the specimen being lighted from all directions at oblique angles, light is projected from only a single azimuth. A variety of illumination scenarios have been employed to provide oblique directional lighting for observation of specimens with the stereomicroscope (an example is presented in Figure 4). Simple diascopic bases are often equipped with a tilting mirror that can be adjusted to provide a certain degree of oblique illumination, but the light is not easily controlled and does not provide a uniform field of view. More complex microscope stands (or bases) have additional control possibilities, including tilting mirrors that are not restricted to a single axis and sliding mirror assemblies that can be inserted and removed from the light path. Some models use one or more sliding baffles to restrict the illumination geometry and ensure that only oblique light strikes the specimen.
Any of these techniques can provide acceptable to excellent results on a wide variety of specimens, but most require a significant degree of manipulation involving the illumination pathway, which is often difficult to quantitatively analyze or reproduce. Involving more art than science, the results achieved with oblique illumination in stereomicroscopy are highly dependent on the microscopist's skill, experience, and patience. A large number of lighting schemes have been developed, which further complicate the issues surrounding this technique for enhancing specimen contrast.
The mechanism by which oblique illumination enhances detail in otherwise almost invisible, colorless specimens is best understood by consideration of the oblique illumination technique as it is commonly described for the classical compound microscope. Direct light from one azimuth of the substage condenser illuminates the specimen from a single side. Oblique illumination is generally achieved by placing a slit or sector stop beneath the lower lens and aperture diaphragm of the condenser, allowing only oblique light passing through the narrow opening of the stop to illuminate the specimen. The effect of the oblique lighting is to shift the zeroth order of light passing through the specimen to the periphery of the objective lens aperture. The shift of the zeroth order to one side enables one or more additional higher orders (sidebands) of the diffracted light to be included at the rear focal plane of the objective and to contribute to the image formation. In many instances, the result is in an increase in optical resolution because the zeroth and some higher orders both contribute to image formation. In addition, the technique also produces an image having shadowed, high relief features that render the specimen pseudo three-dimensional in appearance.
When compared to the darkfield technique, where the specimen is illuminated from all azimuths with highly oblique light, asymmetrical oblique illumination produces images whose character is highly dependent upon the incident angle of illumination. Images produced by oblique illumination are asymmetrical in the sense that edges lying perpendicular to the direction of incident illumination are made visible, while those that lie parallel (or close) to this direction are not. This concept is illustrated in Figure 5 for two specimens at different orientations with respect to the angle of incident oblique illumination. The specimen in Figures 5(a) and 5(b) consists of identical viewfields obtained from a thin single-crystal wafer of lanthanum aluminate, a perovskite that is commonly employed as a substrate for epitaxial thin film deposition of high-temperature superconducting ceramics. Twinning in these crystals hampers confluent thin film formation and can have detrimental effects on the properties of resulting films. The digital image presented in Figure 5(a) depicts the pseudo-relief generated by twinning domains when the crystal is oriented with the longitudinal twin axis parallel to the oblique incident light rays. In contrast, when the crystal (and twin) axis is rotated by 90 degrees, so that it is perpendicular to the incident rays (Figure 5(b)), the twinning domains become readily apparent. This represents a spectacular display of specimen orientation restrictions on the textural effects observed under oblique illumination.
A similar, but less dramatic, result is obtained when observing semi-transparent goat hair fibers with oblique illumination at several orientation angles. When the long axis of the rod-like hair strands is oriented parallel to the incident oblique illumination (Figure 5(c)), structural details in the central portion and edges of the hair fibers is revealed. This detail is absent when the hair fibers are oriented perpendicular to the illumination axis (Figure 5(d)), and a significant difference is observed in the apparent thickness of the fibers between the two orientation angles. Fibers oriented parallel to the incident illumination appear to be much thicker than those oriented perpendicular to the light source. Thus, it is evident that the oblique illumination technique cannot be reliably employed to generate faithful measurement data from images gathered by this method.
The apparent three-dimensional effect afforded by oblique illumination techniques does not represent the actual specimen geometry or topography, and should not be employed to conduct measurements of specimen dimensions. The true value of the oblique illumination image is in revealing transitions in refractive index or other optical path differences within the specimen that enable the morphology and internal structural arrangement to be more clearly understood. The technique can be applied to a variety of materials that appear nearly invisible or transparent in brightfield illumination and cannot be stained or otherwise chemically or thermally treated to enhance contrast. Study of living organisms and processes such as in vitro fertilization, glass or acrylic fibers, chemical crystals, and other unstained materials can be facilitated by the utilization of an easily controlled oblique illumination system.
William Chambers - Industrial Microscope Division, Nikon Instruments Inc., Melville, New York 11747.
Thomas J. Fellers and Michael W. Davidson - National High Magnetic Field Laboratory, 1800 East Paul Dirac Dr., The Florida State University, Tallahassee, Florida, 32310.