Results of VPDC

Copyright: Jugend forscht

Timm Piperīs
microcopy site
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
Summarizing remarks
and conclusions
Video Downloads
List of Personal 
About me
Contact Details

When compared with standard illumination techniques (bright- and darkfield, phase and interference contrast), VPDC is also characterized by a significant increase of axial resolution so that the focal depth (depth of field) is enhanced. Especially in thick and complex textured specimens, the three-dimensional architecture, small internal structures and fine textures at the specimenīs surface can often be visualized better than in concurrent methods. Although the axial resolution is enhanced, the lateral resolution is not visibly diminished. In low density specimens (so-called phase specimens), existing low differences in the optical density can also be visualized by VPDC in a phase contrast-like manner, but haloing can be reduced, and more fine details can be detected in suitable specimens because of the additional darkfield component. In high density specimens (so-called absorption specimens), however, the natural color and existing fine details can appear in higher clarity than achievable by use of brightfield, phase and interference contrast. The Figures 29-34 demonstrate the high grade of image quality achievable with concentric-peripheral VPDC; corresponding examples for axial VPDC are given in the Figurs 35-38.

Fig. 29
gives an overview of the variance in contrast and background brightness achievable with VPDC (Fig. 29b-d) when compared with normal phase contrast (Fig. 29a) and darkfield illumination (Fig. 29e). Darkfield-dominated VPDC-images as shown in Fig. 29d can be transformed into a intermediate and equalized phase-darkfield image (Fig. 29c), when the condenser aperture diaphragm is used for narrowing the external darkfield light annulus in tiny steps. The so-achieved balanced phase-darkfield image according to Fig. 29c can be progresively turned into a phase contrast-dominated VPDC image (Fig. 29b) and finally transformed into a pure phase contrast-like image according to Fig. 29a when the external light annulus is completely covered by the condenser aperture diaphragm.

Fig. 29: Diatom (diameter: 0.12 mm), phase contrast (a), VPDC (b-d), phase contrast-dominated (b), equalized, medium background brightness (c), darkfield-dominated (d), standard darkfield (e)

Fig. 30 demonstrates the high grade of visual information in VPDC (Fig. 30d and e) when compared with standard brightfield (Fig. 30a), phase contrast (Fig. 30b) and darkfield illumination (Fig. 30c). The filamentous algae shown here consist of light absorbing pigmentations and colorless cell structures; they are surrounded by several smaller sized low density adherent material. In brightfield, only the light absorbing components are visualized. In phase contrast, the marginal sessile colorless phase structures are clearly visible, but they are surrounded by small haloing and the higher density zones inside the algae themselves appear very dark so that their structures cannot be perceived in a satisfactory manner. In darkfield, the algae are visible in their natural color and especially the light reflecting internal structures inside them are visualized in high clarity, but some fine details are lost because of blooming or scattering and some adherent material cannot be clearly seen as it is out of focus or not hit by the oblique illuminating light beams.  By use of VPDC, the characteristic advantages of brightfield, phase contrast and darkfield are combined with each other but their particular disadvantages need no longer be tolerated. All in all, the visual information is higher than in the concurrent traditional techniques.

Fig. 30: Filamentous algae (diameter: 0.05 mm), brightfield (a), phase contrast (b), darkfield (c),
VPDC, phase contrast-dominated (d), equalized, medium background brightness (e)

A complementary expressive result is given in Fig. 31. Also in the images shown here, superimposing two phase-contrast- and darkfield-like images leads to enhanced global visual information because low density phase structures remain clearly visible in VPDC and high density structures with a greater thickness appearing black or very dark in normal phase contrast or brightfield are contrasted in their intrinsic color. In brightfiled (Fig. 31a ), the high density Haematococcus pluvialis algae appear like dark silhouettes and the surrounding low density filamentous algae are barely visible. Darkfield illumination (Fig. 31b ) mainly visualises the optically dense Haematococcus pluvialis algae which appear in natural red color. In phase contrast (Fig. 31c), mainly optically thin filamentous algae can be distinguished, wehereas the Haematococcus algae appear dark. The superimposition of phase contrast and darkfield images of this specimen (Fig. 31d) provides additional overall information in one image because both species of algae are clearly revealed in this way.

Fig. 31: Red colored algae (Haematococcus pluvialis), surrounded by low density blue-green algae and detritus,
native preparation, HFW: 0.8 mm, objective 10x, brightfield (a), darkfield (b), phase contrast (c), VPDC (d)

The membranous wing of a beetle shown in Fig. 32 can be presented in dramatically improved clarity when VPDC is carried out. The three-dimensionality and plasticity of the wing is much better visualized in VPDC because the specimen is simultaneously illuminated at different angles and the focal depth is enhanced. Moreover, brightness and contrast are much more balanced and equalized; blooming, scattering and haloing are strongly reduced. When compared with phase contrast (Fig. 32a) and darkfield (Fig. 32b), the global visual information is significantly increased in VPDC (Fig. 32c). In particular, the ribs shows much more fine details and also the small hairs are presented with greater clarity without relevant blooming, scattering or haloing.

Fig. 32: Wing of a beetle (horizontal field width: 1.0 mm), phase contrast (a), darkfield (b), VPDC (c)

In Fig. 33, a marine bristle worm (Polychaeta) from the northern intertidal flats is presented in phase contrast (Fig. 33a), darkfield (Fig. 33b) and VPDC (Fig. 33c). In phase contrast, the wormīs low density soft tissue is well contrasted, but the multiple finer bristles are just barely visible and look like silhouettes. In contrast, the bristles are accentuated in high contrast when illuminated in darkfield, whereas the surrounding soft tissues are not visualized. In VPDC, the setae are visualized as well as the neighboring soft tissue so that the visual information of the phase contrast image is combined with that of the darkfield image. Thus, for example, the roots of the fine bristles in the soft tissues can be seen more easily.

Fig. 33: Marine bristle worm, Polychaeta, (horizontal field width: 1.0 mm), phase contrast (a), darkfield (b), VPDC (c)

Fig. 34 gives an example of biclor light filtering in VPDC based on additional brightfield illumination achieved by a moderate off-centered position of the condenserīs light annuli ("triplex mode"). Phase contrast and bright-field illumination are carried out with red, darkfield illumination with blue filtered light. In normal phase contrast (Fig. 34a), some parts of the crystal are contrasted in black or dark whereas other parts are contrasted in a bright appearance. In the dark and bright zones, fine details are barely visible. In darkfield (Fig. 34b), several contours are irradiated so that they appear in high contrast, whereas other parts of the specimen which are not hit by the oblique illuminating light remain dark so that they cannot be well perceived. In VPDC (Fig. 34c), the concentric light annuli shifted into the condenser are slightly turned into an off centered position so that the specimen is additionally illuminated in a brightfield-like manner. In this variant, the blue filtered darkfield image is superimposed with a red filtered brightfield-like image which is added to red filtered phase contrast illumination. As a result of this technique, the complex structure of the crystal can be visualized in best clarity.

Fig. 34: Crystallization of ascorbic acid (horizontal field width: 1.0 mm), phase contrast filtered in red (a),
darkfield filtered in blue (b), VPDC in bicolor double contrast, light annuli in moderate off-centered position,
additional superposition of a brightfield-like partial image (c)

Also in axial VPDC, several specimens can be visualized in high definition quality even when corresponding standard methods do not lead to adequate results. Fig. 35 gives an example for this. The lamellate structures of the fish scale shown here are just barely visible in brightfield because their optical density is very low and only minimally different from the refractive index of the embedding medium (Fig. 35a). In normal darkfield, only artificial precipitations (some detritus, dust and other particles) are highly contrasted whereas the structure of the scale is nearly invisible (Fig. 35b). Normal phase contrast leads to poor images because the thickness of the preparation is beyond the critical limit and the cover slip of this permanent slide is somewhat inclined (Fig. 35c). In axial VPDC, the scale appears in excellent contrast and clarity, in phase contrast- dominated illumination (Fig. 35d) as well as in equalized (Fig. 35e) and darkfield-dominated APDC (Fig. 35f). In particular, the small marginal denticular stings are accentuated by the axial darkfield image added. Moreover, the depth of field is significantly enhanced when compared with normal darkfield, phase contrast or brightfield illumination.

Fig. 35: Scale of a Silver Angelfish (Pterophyllum scalare), permanent slide, cover slip preparation,
horizontal field width (HFW): 0.16 mm, brightfield (a), darkfield (b), normal phase contrast (c),
axial VPDC (d-f), dominance of phase contrast (d), equalized image (e), dominance of axial darkfield (f)

Also high density specimens which are not well suited for phase contrast can be successfully examined in axial VPDC. In Fig. 36, different weightings of phase contrast and axial darkfield are demonstrated with rather thick diatoms showing fine superficial textures. It is obvious that the depth of field is significantly increased and additional fine pores in the depicted casings are much more clearly discernible when the aperture diaphragm of the condenser is narrowed in small increments. Phase contrast dominates when the aperture diaphragm is fully open (Fig. 36a and d). Moderate closure of the aperture diaphragm adds axial darkfield illumination so that, in particular, fine openings on the surfaces are visualised with much greater clarity (Fig. 36b and e). Closing the aperture diaphragm even further highlights, in particular, the finely porous openings in the diatom shells with a maximum contrast (Fig. 36c and f).

Fig. 36: Diatoms, arranged slide, high density frustules, HFW: 0.10 mm, phase contrast (a, d),
axial VPDC, dominance of phase contrast (b, e) and axial darkfield (c, f)

Also in flat and rather low density phase specimens, axial VPDC can lead to higher sharpness of contours and improved distinctness of fine linear structures when haloing is reduced; examples are given in Fig. 37a and b.

Fig. 37: Low density diatoms, arranged slide, HFW: 0.17 mm, phase contrast (a), axial VPDC (b)

As depicted in Fig. 38, it can be beneficial to use bicolor contrast with axial VPDC if the visual information provided by darkfield and phase contrast is highlighted in different colors. In this example, the axial light leading to axial darkfield is filtered in red and the phase contrast producing light annulus is fitted with an annular blue filter. Fine crystalline structures (shown in red in axial darkfield) are highlighted in red, particularly the filigree details on the surface of the microscope slide and in the depicted main crystal (Fig. 38a). The three -dimensional architecture of the pyramidal main crystal is accentuated by the blue filtered phase contrast partial image. Incremental closure of the aperture diaphragm gradually decreases the fraction of phase contrast-generating light so that the structures illuminated in axial light appear bright on a dark background, thus emphasising them even further (Fig. 38b).

Fig. 38: Pyramidal alum crystallization, HFW: 0.8 mm, axial VPDC in bicolour double contrast,
axial light filtered in red, phase contrast producing background light filtered in blue,
equalized superposition of axial darkfield and phase contrast (a), dominance of axial darkfield (b)

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