Principles of VPBC

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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
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Further developments
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In VPBC, the specimen is simultaneously illuminated in phase contrast and brightfield each filtered in different colors. Moreover, the illuminating light beams leading to brightfield and phase contrast are separated from each other and also different with regard to their intensities and angles of incidence. In this way, two partial images are optically superimposed and interfere with each other – one phase contrast and one brightfield image. The brightness of both different images and so the weighting of the phase contrast and brightfield illumination can be regulated by the user in tiny steps. Thus, the appearance of resulting images can be modulated from phase contrast-dominated or equalized (balanced) to brightfield dominated images.

In standard mode, phase contrast and brightfield images are both generated on the basis of concentric (coaxial, azimuthal) illumination. Eccentric (oblique) illumination can be achieved when parts of the illuminating light beams are covered by a facultative light stop which can be integrated into the illuminating light path. In phase contrast, the specimen is illuminated by a light cone in the same manner as is usual in standard technique. Brightfield illumination can be generated based on axial or peripheral light. In the latter case, phase contrast is combined with circular oblique lighting (COL, James, 2012).

By the optical means described, high density light absorbing structures and low density phase shifting components can be simultaneously visualized. As the illuminating light components leading to brightfield and phase contrast are filtered at different colors, and as they are separated from each other and run to the specimen at different angles of incidence, the phase contrast image is not “disturbed“ by the brightfield image superimposed and the clarity of the brightfield image is not reduced by the phase contrast image simultaneously generated. Halo artefacts and shade-off are significantly reduced, so that fine details and marginal contours are visualized with greater precision even in low density phase specimens. In thick specimens, the accuracy of fine and small structures can be significantly enhanced by use of VPBC when compared with standard applications in phase contrast or brightfield. In thin specimens, additional contrast effects can be obtained when both partial images interfere with each other. The 3D-appearance and the plasticity of the specimen can be improved and the depth of focus enhanced, because the aperture diaphragm can be used for modulations of the imageīs appearance and the illuminating light components associated with brightfield and phase contrast are different with regard to their angles of incidence.

Optical solutions for VPBC

VPBC can be carried out with normal phase contrast objectives equipped with a phase plate and a phase ring. The light mask within a phase contrast condenser fitted with an appropriately sized light annulus for phase contrast illumination has to be modified for VPBC so that an additional brightfield image is generated based on axial or concentric peripheral light. For axial brightfield illumination (axial VPBC), a small centric perforation has to be added in the middle (center point) of the light annulus; this perforation is congruent with the optical axis. Alternatively, the light mask can be fitted with an additional larger sized peripheral (external) light annulus which is concentric with the smaller internal light annulus for phase contrast. Instead of transparent light segments, the external light outlet can also consist of small perforations. The illuminating light pathway for axial and peripheral VPBC is schematically shown in Fig. 14.

Fig. 14:
athway of the illuminating light for axial (a) and peripheral (b) VPBC.

1 = light source
2 = modified light mask fitted with a couple of separate concentric light outlets for phase contrast and brightfield
3 = condenser lens
4 = specimen slide
5 = objective lens
6 = phase plate with phase ring
7 = illuminating light used for phase contrast
8 = illuminating light used for axial (8a) and peripheral (8b) brightfield
9 = eyepiece with intermediate image
10 = eye


The imaging light which is diffracted by the specimen runs the same way as in normal brightfield and phase contrast and is therefore not shown in these light paths. Prototypes of light masks designed for axial and peripheral VPBC are presented in Fig. 15. In the examples shown here, the phase contrast producing light annuli are filtered in blue and the light outlets necessary for simultaneous brightfield illumination are filtered in red. It is essential for well balanced results that the areas of the light outlets associated with phase contrast and brightfield are similar or equal so that the intensities of the respective partial images are well balanced. The proper alignment of so-modified light masks can be visually controlled by use of a phase telescope in the same manner as is usual in normal phase contrast techniques.

Fig. 15:
Prototypes of condenser light masks for axial (a) and peripheral (b) VPBC, mounted on slides, light annuli for phase contrast filtered in blue, outlets for brightfield filtered in red.


Fig. 16 gives examples for correct alignments of the light masks for axial and peripheral VPBC shown in Fig. 15. In both variants - axial and peripheral VPBC -, the light outlet which is situated in a peripheral position can be influenced by the condenser aperture diaphragm so that the intensity of the corresponding illuminating light can be reduced by closing this diaphragm. Dependent on the type of light mask used, the intensity of the brightfield or phase contrast partial image can be regulated by the aperture diaphragm in tiny steps so that the resulting composite image may be equalized and balanced, or dominated, by phase contrast or brightfield. When the diameter of the aperture diaphragm is moderately reduced, depth of field, global contrast and accentuation of marginal contours can also be enhanced - comparable with normal brightfield examinations. To obtain oblique illumination, a light stop can be slit into the condenser as mentioned above so that parts of the illumination light are blocked. Alternatively, the annular light outlets shown in the Figures 14-16 could be replaced by segmental arched perforations.

Fig. 16: Alignment of the light masks shown in Fig. 15, images taken from a phase telescope, axial (a) and peripheral (b) VPBC.


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