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.
Pathway 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
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)
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.
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