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Introduction In common dark field illumination, the condenser either is not equipped with an aperture diaphragm, or an existing condenser diaphragm has to remain in wide position. Thus, the focal depth is lower than in usual bright field images. Moreover, dark field imaging is associated with marginal blooming, especially in linear structures exhibiting with large differences in phase or density (e.g. cell walls, edges in crystals and other mineralogical material). As specimens are illuminated by oblique light that comes from the periphery of the illuminating apparatus, fine structures inside them may only be barely or sometimes not visible. Last, dark field images are characterized by a high dynamic range, i.e. strong differences in brightness and a very high contrast between dark background and bright specimens. Thus, several modifications of existing optical equipment have been made in order to achieve fundamental improvements of the global image quality in darkfield imaging especially in observations of three-dimensional specimens.
Implementation of “condenser aperture reduction darkfield“ In the optical arrangement designed for condenser aperture reduction phase contrast (see separate description), the small condenser annulus necessary for phase contrast illumination is replaced by a greater annulus so that the inner diameter (ri) of the respective condenser annulus is just a little greater than the diameter of the object field or the optical diameter of the respective objective (ro). The principle of this arrangement is shown in fig. 1.
Fig. 1: Achievement of condenser aperture reduction darkfield
When the light ring is adjusted in centered position (fig. 1b), a black background can be achieved, and the specimen is illuminated in a concentric circular manner (360°). When the light ring is moved to an off-centered position by a tiny shift (fig. 1c), oblique illumination effects can be achieved so that the three-dimensional relief of the specimen is accentuated, while the background is moderately brightened. Summing up, two illumination modes can be achieved in this way: concentric and eccentric condenser aperture reduction darkfield. In the same manner as in aperture reduction phase contrast, the condenser aperture diaphragm is projected into a medium position situated between the specimen plane and the objective´s back focal plane. Moreover, the focal intercept of the condenser has to be be modified so that it will be circa 11mm lower than in usual techniques. By changing the focal intercept, the illuminating light can run at steeper angles so that the specimen can be illuminated more centrically in an axial direction. As a result of these modifications in the optical design, the illuminating light running through the light ring within the condenser is no longer stopped when the condenser aperture diaphragm is closed. Therefore, the visible depth of field can be significantly enhanced by closing the condenser diaphragm. When the objective is fitted with an additional integrated iris diaphragm mounted in its back focal plane, the resulting focal depth can be enhanced even more as the projection plane of the condenser diaphragm is different from the plane of the objective´s diaphragm. Because of its aberrant position, the condenser iris diaphragm can also work in a similar way as the usual field diaphragm. Moreover, the illuminating light path is modified with regard to the angle of incidence, because the focal intercept of the universal condenser is circa 11 mm lower than it would be using the original condenser. In conventional darkfield, the illuminating light beams travel to the specimen from the periphery so that the specimen is illuminated in an oblique manner. In condenser aperture reduction darkfield, the illuminating light travels at a higher angle so that the specimen is more centrically illuminated. As aperture reduction darkfield is carried out in transmitted light, it can be combined with darkfield techniques based on epi -illumination (superimposed darkfield in sandwich illumination). Of course, a suitable illuminator for epi-illumination and special objectives designed for darkfield examinations in incident light have to be availiabe for this task (fig. 2). An industrial microscope, constructed for wafer inspections and designed for various illumination techniques in epi-illumination and transmitted light, was used for these optical experiments..
Fig. 2: Optical pathway in darkfield based on epi-illumination,
In asandwich illumination, the visual information can be accentuated still more, when both components of the illuminating light are filtered in different colors. By use of monochromatic narrow band filters, the resolution and sharpness of the resulting image can be optimized more - up to the physical limit of the respective optical system. In some specimens, fine superficial structures can also impressively be documented in darkfield when epi-illumination carried out so that the specimen is solely illuminated by incident light. In digital photomicrographs, the appearance of the background can be modified by postprocessing. Thus, for instance, a dark or black background can be brightened or transformed into an aberrant color. This modification can lead to very impressive presentations, in most cases superior to the corresponding original material.
Results By “condenser aperture reduction dark field“, the visible depth of field can significantly be enhanced in life observations as well as in photomicrography when compared with traditional darkfield illumination. Marginal blooming which appears as a typical artifact in most darkfield images can be mitigated or completely eliminated by closing the condenser iris diaphragm. Peripheral scattered light components are reduced more rigorously than achievable by closing a usual field diaphragm. THus, the condenser aperture diaphragm works in double mode standing alone as aperture and field diaphragm. When the objective is fitted out with an integrated iris diaphragm typically mounted in the back focal plane, the depth of field can be enhanced even more. The three-dimensional relief of some specimens can be visualized in an improved manner when condenser aperture reduction darkfield is carried out in oblique illumination technique (eccentric variant). Fig. 3-6 demonstrate all relevant optical effects which are achievable by the various modifications of “condenser aperture reduction darkfield“ described above. When darkfield is obtained by epi-illumination (see fig. 2), fine superficial structures in some transparent or semi-transparent specimens may also be visible in high clarity (fig. 7 ). Existing details in texture can be accentuated even more, when darkfield in epi -illumination is combined with condenser aperture reduction darkfield illumination in transmitted light (“superimposed darkfield“,). In this case, additional improving effects can be achieved when both illuminating light components are filtered in different colors, preferably by use of monochromatic narrow band filters (fig. 8-10).
Fig. 3: Radiolarians, horizontal field width: 0.8 mm,
Fig. 4: Radiolarian (length: 0.24 mm),
Fig. 5: Radiolarian (diameter: 0.26 mm),
Fig 6: Radiolarian (diameter: 0.26 mm),
Fig. 7: Radiolarian (length: 0.28 mm), darkfield illumination with incident light
Fig. 8: Radiolarian (diameter: 0.30 mm),
Fig. 9: Diatoms, (diameter or length: 0.12 mm), sandwich illumination,
Fig. 10: Diatom (Diameter: 0.14 mm), sandwich illumination,
Publications Piper, J.: Improved imaging of three-dimensional transparent specimens in modified darkfield techniques and digitized interference contrast (in German) Piper, J.: Improved techniques for imaging of three-dimensional transparent specimens in advanced darkfield and interference contrast modes.
Copyright: Joerg Piper, Bad Bertrich, Germany, 2010
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