When compared with brightfield,
darkfield and phase contrast illumination carried out stand-alone, much more details can be perceived by VPBC, especially in complex structured specimens consisting of high density
light absorbing and low density phase shifting details. The complementary characteristic visual information which is apparent in brightfield and phase contrast is combined in the resulting VPBC images. As the final
image can be continuously modulated from a phase contrast-dominated to a
brightfield-dominated character, the illumination can be adapted to the average or prevalent optical density of the specimen.
The three-dimensional architecture in thick transparent specimens can often be visualized better when compared with standard techniques as well as small internal structures and fine textures within thin
and low density specimens. Although the axial resolution is enhanced, the lateral resolution is not visibly diminished. When compared with standard phase contrast, haloing and shade-off are significantly reduced. In
thin parts of colorless specimens, the differently colorized brightfield and phase contrast images can interfere with each other so that additional color contrast effects can
sometimes be apparent. Thus, for instance, fine
details can be optically contrasted in green color tones even when the illuminating light components are filtered in red and blue.
Photomicrographs taken in VPBC are well suited for converting to black and white. In particular, all immanent fine details and structures can be highly accentuated when the color channels for red and blue
are separately regulated with regard to their dominance and gradation while the conversion into B&W is carried out.
The high grade of visual information which can be obtained by
VPBC is demonstrated in the Figures 39-44. Examples for axial VPBC are given in the
Figures 39-42, practical results achieved with peripheral VPBC are presented in the Figures 43 and 44.
Fig. 39 shows a colorless crystallization (ascorbic acid) prepared without cover slip and consisting of thick multilayered crystals, surrounded by thin and low density precipitations. In
brightfield (Fig. 39a), only the thick crystals are visible in a satisfying quality. In phase contrast (Fig. 39b), the surrounding precipitations are contrasted with intensive halo artefacts,
whereas the structures within the neighboring crystals are imaged in reduced clarity because of haloing and critical thickness. In axial VPBC (Fig. 39c), the thick crystals are visualized with maximum clarity
as well as the low density flat precipitations. The marginal contours of the flat phase shifting precipitations can be recognized with greatest precision, because haloing is strongly reduced.
Fig. 39: Ascorbic acid (horizontal field width: 0.3 mm), objective 20x, NA 0.32,
brightfield (a), phase contrast (b),
In Fig. 40, detail views of Fig. 39 are presented in Black and White. The intensity and gradation of the blue and red color channels were
manually adjusted when the conversion was carried out. It can be demonstrated that
VPBC can also lead to the best results in B&W imagery.
Fig. 40: Detail view from Fig. 39 (horizontal field width: 0.24 mm), conversion into B&W, equipment from Fig. 39
A frustule of a pennate diatom (Pinnularia sp.) is shown in Fig.
41 (total view) and Fig. 42 (detail view), taken in
brightfield (a), darkfield (b), phase contrast (c) and axial VPBC (d). In this specimen, the lamellate structures appear with highest accuracy when VPBC is
carried out. Moreover, the axial resolution (depth of field) is significantly enhanced in VPBC so that a maximum vertical distinctness can be achieved.
Fig. 41: Diatom (Pinnularia sp.), total length: 0.275 mm, objective 32x, NA 0.40,
brightfield (a), darkfield (b), phase contrast (c), axial VPBC
Fig. 42: Detail view from Fig. 41 (horizontal field width: 0.12 mm), equipment from Fig.
distances of lamellae: circa 2 ĩm
Fig. 43 shows colorless alum crystallizations prepared without cover slip and characterized by variable thickness and multiple fine internal details. Single images were taken in
brightfield (Fig. 43a
), darkfield (Fig. 43b), phase contrast (Fig. 43c) and concentric peripheral VPBC (Fig. 43d
). In VPBC, the fine details inside the crystals are contrasted with greatest clarity as are thin-layer
precipitations which can be discriminated from the background with highest accuracy and precision when examined in VPBC.
Fig. 43: Alum crystallization (horizontal field width: 0.3 mm), objective 10x, NA 0.25,
darkfield (b), phase contrast (c), concentric-peripheral VPBC
Fig. 44 gives an example of a discoid diatom (Coscinodiscus sp.)
taken in standard techniques (Fig. 44a-c) and peripheral VPBC (Fig. 44d
). Also in this specimen, fine structures are accentuated with highest clarity when observed in VPBC. In
(Fig.44a), the global contrast is poor and low density structures are lost, in
Fig. 44b), fine structures are irradiated because of blooming and scattering, and in normal phase contrast (Fig. 44c), marginal contours are affected with haloing. In both
darkfield and phase
contrast, the vertical resolution is limited because the aperture diaphragm
- if present - has to remain wide open. In VPBC, all contours of fine details
can be clearly perceived without being masked by haloing, blooming or scattering and the vertical resolution is maximized.
Fig. 44: Diatom frustules, center: Coscinodiscus sp. (horizontal field width: 0.264 mm), objective 32x, NA 0.40,
brightfield (a), darkfield (b), phase contrast (c), concentric-peripheral
Last Update: August 10th, 2012
Copyright: Timm Piper, 2012