Darkfield observation in stereomicroscopy requires a specialized stand containing a reflection mirror and light-shielding plate to direct an inverted hollow cone of illumination towards the specimen at oblique angles. The principal elements of darkfield illumination are the same for both stereomicroscopes and more conventional compound microscopes, which often are equipped with complex multi-lens condenser systems or condensers having specialized internal mirrors containing reflecting surfaces oriented at specific geometries.
Darkfield microscopy is a simple and popular method for rendering unstained and transparent specimens clearly visible. Good candidates for darkfield observation often have refractive indices very close in value to that of their surroundings and are difficult to image with conventional brightfield techniques. As an example, small aquatic organisms, oocytes, and cells in tissue culture have a refractive index ranging from 1.2 to 1.4, resulting in a negligible optical difference from the surrounding aqueous medium (refractive index of 1.3). These and similar specimens are ideal candidates for observation with darkfield illumination techniques.
The configuration presented in Figure 1 illustrates a modern Nikon SMZ1500 stereomicroscope equipped with an advanced stand containing provisions for both brightfield and darkfield illumination through a clear glass stage mounted on the top of the stand. Also depicted is a digital Internet camera system (Nikon Dn100) capable of transferring images collected by the microscope to remote observers. Details of the darkfield illumination mechanism are discussed below.
Illumination of specimens by darkfield requires blocking out of the central light rays along the optical axis of the microscope, which ordinarily pass through and around (surrounding) the specimen. Blocking these light rays allows only those oblique rays originating at large angles to strike the specimen positioned on the microscope stage. In a compound microscope equipped with a simple condenser system, the condenser (Abbe-style) top lens is spherically concave, enabling light rays emerging from the surface in all azimuths to form an inverted hollow cone of illumination having an apex centered in the specimen plane. If no specimen is present on the stage, and the numerical aperture of the condenser is greater than that of the objective, the oblique rays cross and miss entering the objective front lens because of their obliquity. The field of view will appear dark.
The stereomicroscope illustrated in Figure 1 produces an oblique cone of illumination using a specially-designed seven-sided toroidal mirror (Figure 2) that substantially reduces the stray light entering the large common main objective front lens. The toroidal mirror operates in a manner similar to high numerical aperture reflecting darkfield condensers that are equipped with internal mirror surfaces having a variety of curvature geometries.
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One of the most popular darkfield condenser designs, heavily utilized for high magnification compound microscopy prior to the emergence of phase contrast, is the paraboloid condenser, which has a curved and mirrored cardioidal internal surface. Illuminating light passes through the condenser and reflects from a single surface that is made from a paraboloid truncated by a light stop oriented perpendicular to the condenser and microscope optical axis. This system is free from spherical, chromatic, and coma aberrations and produces a sharply focused cone of illumination for the specimen from all azimuths. Although the stereomicroscope toroidal mirror design illustrated in Figures 2 and 3 does not operate with the sophistication and precision of the paraboloid condenser, it is far more effective for illuminating specimens in darkfield than conventional reflection mirrors that have a cylindrical geometry. The diagrams in Figure 3 compare the toroidal mirror design with a more conventional cylindrical mirror found in a majority of stereomicroscopes. In addition to providing more even illumination from all azimuths, the toroidal condenser design substantially reduces the amount of stray light entering the objective front lens, which leads to a significant enhancement of contrast between the specimen and background.
When a transparent specimen is placed on the glass microscope stage and observed under darkfield illumination, the oblique light rays cross the specimen and are diffracted, reflected, and/or refracted by optical discontinuities (such as the cell membrane, nucleus, and internal organelles) allowing these faint rays to enter the objective. The specimen then appears bright on an otherwise black background. In terms of Fourier optics, darkfield illumination removes the zeroth order (unscattered light) from the diffraction pattern formed at the rear focal plane of the objective. This results in an image formed exclusively from higher order diffraction intensities scattered by the specimen, and is also responsible for the main limitation of darkfield observation. Because the image is composed entirely from scattered light from the specimen, it is rich in glare and can even be distorted to varying degrees, so it cannot be considered a faithful geometrical reproduction of the specimen.
The digital images in Figure 4 illustrate the effects of darkfield and brightfield illumination on fibers in whole mount specimens prepared using Canada balsam and a microscope slide and coverslip. Figure 4(a) and 4(b) compare nylon fibers under conditions of brightfield (Figure 4(a)) and darkfield (Figure 4(b)) illumination. The fibers imaged with brightfield are seriously lacking in contrast and minute details are difficult to distinguish against the white background. In contrast, when the fibers are illuminated with darkfield techniques (Nikon SMZ1500 with a toroidal mirror illuminator), internal fiber detail is discernable to a higher degree and depth of field emphasis becomes more pronounced. A situation where fibers have too much contrast in brightfield is presented in Figure 4(c) for pineapple fibers, which are not transparent and almost opaque when visualized under brightfield illumination. Viewing the same pineapple fiber specimen with darkfield illumination reveals far more intricate detail (Figure 4(d)) and exposes longitudinal splits in the fibers that are not apparent in the brightfield image.
Specimens that have smooth reflective surfaces produce darkfield images that are primarily due to reflection of light into the objective. In situations where the specimen refractive index is different from the surrounding medium or where refractive index gradients occur (as in the edge of a membrane), light is refracted by the specimen. Both instances of reflection and refraction produce relatively small angular changes in the direction of light, enabling some rays to enter the objective. In contrast, some light striking the specimen is also diffracted, producing a 180-degree arc of light that passes through the entire numerical aperture range of the objective. The resolving power of the objective is the same in darkfield illumination as that achieved under brightfield conditions, but the optical character of the image (as discussed above) is not as accurately reproduced.
In darkfield microscopy, contrast is greatly enhanced by the superposition of a brightly shining specimen on a dark background. Blocking of zeroth order light rays by an opaque stop enables only higher order light rays to bathe the specimen with illumination. Highly oblique light rays, diffracted by the specimen and yielding first, second, and higher diffracted orders at the rear focal plane of the objective, proceed onto the image plane where they interfere with one another to produce an image of the specimen.
If the rear of the objective in a stereomicroscope operating in darkfield illumination is viewed using a Bertrand lens or eyepiece telescope, it will appear filled with light. The faint diffracted light is reconstituted into a visible image at the plane of the eyepiece diaphragm with its contrast reversed to produce a bright image on a dark background. Because darkfield microscopy eliminates the bright, undiffracted zeroth order light, this form of illumination is very wasteful of light and thus demands a high intensity illumination source. Stereomicroscope illumination stands that are equipped for darkfield illumination take this factor into account, and high-intensity tungsten halogen bulbs are provided to produce sufficient light flux for the purpose.
A number of aftermarket products are currently available for retrofitting stereomicroscopes with transmitted darkfield illumination. In addition, many of the microscope manufacturers offer illumination accessories that can be conveniently utilized to achieve darkfield conditions for their stereo systems. Typical aftermarket darkfield illuminators are presented in Figures 5 and 6. The design illustrated in Figure 5 utilizes a fiber optic ring light to provide illumination for a specially crafted stage that contains an internal mirror system and an opaque light stop. Light from the ring light illuminator is reflected from the internal cylindrical mirror with the central (zeroth order) rays being blocked by the light stop to form an inverted cone of illumination. Specimens are placed directly onto a glass plate resting above the stage aperture and can then be visualized with darkfield illumination. The ring light is equipped with an external light source that contains a voltage supply and a high-intensity tungsten-halogen lamp. Another darkfield condenser design, which also contains provisions for brightfield illumination, is presented in Figure 6. This condenser system utilizes a slider to rotate between brightfield and darkfield illumination and also contains a light source coupled to the condenser by a fiber optic bundle.
Ideal candidates for darkfield illumination in stereomicroscopy include minute living aquatic organisms, diatoms, small insects, bone, fibers, hair, unstained bacteria, yeast, and protozoa. Non-biological specimens include minerals, chemical crystals, colloidal particles, inclusions and porosity in glass, ceramics, polymer thin sections, and refractive index gradients. Care should be taken in preparing specimens for darkfield microscopy because features that lie above and below the plane of focus, especially fingerprints, dust, fibers, and cleaning residue, can also scatter light and contribute to image degradation. Specimen thickness and microscope slide thickness are also very important and, in general, a thin specimen is desirable to eliminate the possibility of diffraction artifacts that can interfere with image formation.
Specimens imaged under the proper conditions of darkfield illumination are quite spectacular in appearance (try, for instance, a drop of fresh blood). Often specimens containing very low inherent contrast in brightfield microscopy are readily observable in darkfield, and this type of illumination is ideal for revealing outlines, edges, boundaries, and refractive index gradients. Unfortunately, darkfield illumination is less useful for revealing internal details. Other types of specimens, including many that have been stained with dyes, also respond well to illumination under darkfield conditions. These include plant and tree thin sections (stained and unstained), diatoms, radiolarians, fossils, bone sections, embryos, and hair (both human and animal).
During the first half of the twentieth century, darkfield microscopy (both compound and stereo) had a very strong following and a great deal of effort was expended in optimizing darkfield condenser systems and illuminators. This intense interest slowly began to fade with the emergence of more advanced contrast-enhancing techniques such as phase contrast, differential interference contrast, and Hoffman modulation contrast. Recently, new stereomicroscope illumination techniques, such as Nikon's oblique coherent contrast, which dramatically increase the contrast of transparent specimens, are being introduced and will ultimately probably displace a significant amount of interest in darkfield stereomicroscopy. However, a renewed interest in transmitted darkfield microscopy has arisen due to its advantage when used in combination with fluorescence microscopy.
Darkfield microscopy is still an excellent tool for both biological and medical investigations. The technique can be effectively utilized to view a wide spectrum of biomedical and industrial specimens and can often reveal details that are not visible with other illumination methodology.
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.