Principles of VBDC

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Principles of VBDC
Principles of VPDC
Principles of VPBC
Materials and Methods
Results of VBDC
Results of VPDC
Results of VPBC
Further developments
Further developments
Further devepolments
Optical calculations
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In VBDC, the specimen is simultaneously illuminated by two different components of transmitted light which create two partial images, one bright- and one darkfield-like image. These partial images are optically superimposed and interfere with each other in live examinations as well as in photomicrographs. The amount or amplitude of the different illumination light components and thus also the intensity and brightness of the partial images can be separately regulated by the user. In this way, the character of optical contrast can be modified in tiny steps, and the appearance of the resulting image can be continuously changed from a bright-field-like aspect via overlapping images with medium background brightness, up to a darkfield-like appearance. Moreover, VBDC can be achieved based on axial, azimutal, concentric, eccentric or oblique illumination, and in the oblique illumination variants, the angle of incidence can be continuously adjusted. In order to achieve VBDC, the condenser has to be designed in a particular manner. A facultative light stop can additionally be integrated into the light path if necessary. In all modifications of VBDC, the condenser aperture diaphragm can be used for improvements of the final image quality on demand.

By the optical means described above, light absorbing and light reflecting components within the specimen are simultaneously visualized. The brightfield like partial image is based on the principal zeroth order maximum and achieved by absorption and diffraction of the transmitted light. In the darkfield-like partial image, the principal zeroth order maximum does not contribute to the resulting image, as the corresponding illuminating light beams run past the objective lens. For this reason, this image is only based on secondary maxima and reflected and scattered light components (Determann and Lepusch, 1981b). The particular contrast effects of my new method result from the superimposition and interference of these completely different partial images.

Optical solutions for VBDC  

Several different optical solutions are suitable for VBDC-imaging. They can be divided into two groups: condenser-based and light stop-based. In both techniques – condenser- and light stop-based -, the specimen can be illuminated by axial / concentric or oblique / eccentric light beams.

Condenser-based concentric VBDC

Fig. 1:
Light pathway for condenser-based concentric (a) and eccentric (b) VBDC,
illuminating light corresponding with brightfield (1) and darkfield (2),
imaging light, bent and reflected by the specimen (3), LA = light annulus.
Condenser lenses are not drawn in the figures.

When concentric VBDC has to be carried out, a standard universal condenser for phase contrast illumination has to be modified as follows: The light annuli within the condenser have to be replaced by a set of appropriately designed larger sized annuli. The inner diameter of the respective light annulus has to be somewhat smaller and its external diameter has to be somewhat larger than the diameter of the corresponding objective´s internal cross section area. In this way, the specimen is concentrically illuminated in a brightfield-like manner by transmitted light beams which come from the internal section of the light annulus and run through the peripheral zones of the objective lenses. Additionally, the specimen is also illuminated in a darkfield-like manner by concentric light beams from the external section of the light annulus which run in oblique direction without reaching the objective lenses. In both so-generated partial images, the specimen is illuminated by a concentric light cone. The corresponding light path is demonstrated in Fig. 1a.

Fig. 2: Optical alignment for cendenser-based VBDC, controlled with a phase telescope,
concentic VBDC with dominance of brightfield (a) and darkfield (b), eccentric VBDC (c)

By use of a phase telescope, the alignment of the light annulus can be visually controlled (Fig. 2a and b). The internal zone of the light annulus is projected into the marginal periphery of the objective´s cross section area, whereas the external zone of the light annulus is not visible as it is situated outside the territory of the objective. When the internal zone of the light annulus is very small, the final image will be dominated by the darkfield component (Fig. 2b), and the bright-field illumination will be dominant when the broadness of the internal illuminating zone is increased (Fig. 2a).

The breadth of the external zone which corresponds with the darkfield-like image can be reduced with the help of the condenser aperture diaphragm if necessary. In this way, the vertical resolution (focal depth) can be enhanced and potential darkfield-associated blooming and scattering can be attenuated. When the objective is fitted with an iris diaphragm mounted in its back focal plane, the objective´s cross section area can be reduced on demand so that it can also be adjusted to rather small light annuli being lower sized than the objective´s cross section.

The projection of the condenser annulus and the run of the illuminating light components can be modified and adjusted in tiny steps when the condenser is shifted in vertical direction. Alternatively, the condenser´s superior lens group could be designed as a Zoom system so that the focal intercept could be continuously changed.

Condenser-based eccentric VBDC:

To obtain an oblique illumination, the condenser annulus has to be moved to an off-center position so that the periphery of the objective´s cross section area and the internal zone of the condenser annulus overlap with each other. The other arrangements are the same as described for concentric VBDC. The light pathway of this mode is shown in Fig. 1b, the corresponding alignment controlled by a phase telescope is presented in Fig. 2c.

Light stop-based axial VBDC:

Fig. 3:
Light stop with twin diaphragm

A high variability in VBDC-imaging can be achieved when a light stop is integrated into the light path. This light stop has to be situated in the back focal plane of the objective or near its back focal plane. In my prototype, I placed the light stop directly above the objective barrel and the revolving nosepiece (objective turret). As shown in Fig. 3, my light stop model was built as a black slide fitted with two circular holes acting as twin diaphragm. When this slide is inserted in an adequate position, the imaging light can pass the twin diaphragm, whereas the illuminating light is partially or completely blocked. In this arrangement, either the black area between both holes or the neighboring areas beside each hole can act as a light stop.

Fig. 4: Light pathway for light stop-based concentric (a) and eccentric (b) VBDC,
light stop adjusted for axial darkfield illumination
illuminating light (1), imaging light, bent and reflected by the specimen (2),
LA = light annulus, LS = light stop, CID = condenser iris diaphragm
Condenser lenses are not drawn in the figures.

As shown in Fig. 4a and 5a, an axial VBDC illumination can be achieved when a large sized condenser annulus is turned into an equatorial and axial position so that the illuminating light is congruent or nearly parallel with the optical axis. By closing the condenser iris diaphragm, the area of the illuminating light zone can be reduced further so that the illuminating light is collimated (Fig. 5b). The light stopping slide has to be inserted into the light path as shown in Fig. 5c. When the illuminating light is completely blocked, an axial darkfield image will result. When the light stop is moderately shifted into a paramedian position, a small part of the illuminating light can run through the specimen and the objective lenses so that a brightfield image is superimposed. The weighting of both partial images (bright and darkfield) can be regulated with the light stop by moderate changing of its position. By shifting the light stop from a median into a progredient paramedian position, the appearance of the resulting image and the brightness of the background can be continuously changed from axial darkfield to VBDC or brightfield illumination. The higher the misalignment of light stop and condenser annulus, the higher the dominance of the bright-field-like image will be. Within the light stopping slide, the transparent hole which is passed by the transmitted illuminating light corresponds with the brightfield-like partial image, and the other hole is preferably to be passed by the reflected and scattered imaging light components which are associated with the darkfield-like partial image.   

Fig. 5: Optical alignment for light stop-based axial VBDC, controlled with a phase telescope,
equatorial position of the light annulus (a), collimation of the illuminating light beams by the
condenser iriis diaphragm (b), light stop in equatorial position (c)

Light stop-based oblique VBDC:

According to the light path shown in fig. 4b, the specimen can be illuminated by an oblique light beam, when the condenser annulus is shifted in lateral direction to a paramedian or paraequatorial position. The light stop has to be shifted in the same direction so that it remains congruent with the illuminating light zone. In this way, the grade of eccentricity and the angle of incidence can be continuously varied. A maximum eccentricity is achieved when the condenser annulus is turned to the marginal periphery of the objective´s cross section area (Fig. 6a). In this position, the illuminating light zone can practically be covered by one of the black areas aside from the slide´s transparent holes (Fig. 6b). In this arrangement, the weighting of the dark- and brightfield illumination can be adjusted and changed when the light stop is moderately shifted. Also in this variant, the illuminating light can be collimated by an adjustable condenser iris diaphragm (Fig. 4b).

Fig. 6: Optical alignment for light stop-based oblique VBDC, controlled with a phase telescope,
light annulus in maximum marginal (lateral) position (a), covered by the light stop (b),
light annulus in concentric postition, covered from one side by the light stop (c)

In concentric VBDC carried out according to Fig. 2a or b, a selective oblique illumination can be achieved for the brightfield partial image when a sector of the illuminating light zone is covered by the light stopping slide shown in Fig. 3 (Fig. 6c). Instead of this, a global oblique illumination can be obtained when a sector of the light annulus is covered in the condenser so that the bright - and darkfield-like partial images are each generated by oblique light.    

Last Update: August 10th, 2012
Copyright: Timm Piper, 2012