When compared with standard illumination techniques (bright and darkfield, phase and interference contrast), VBDC is characterized by a significant increase of axial resolution so that the focal depth
(depth of field) is significantly enhanced. Especially in thick and complex textured specimens, the three-dimensional architecture can be visualized much better than in concurrent methods. Although axial resolution
is enhanced, lateral resolution is not diminished in a visible manner. Also small structures inside transparent specimens, fine textures at the specimenīs surface and three-dimensional reliefs are often visualized
with superior clarity. In low density specimens, existing low differences in the optical density can also be visualized by VBDC in a phase or interference contrast-like manner. The high grade of visual information
which can be obtained by VBDC is demonstrated in the Figures
Fig. 21 demonstrates the high grade of visual information in concentric condenser-based VBDC (Fig. 21b and c) when compared with
standard bright- and darkfield illumination (Fig. 21a and d). The varnish
(Entellan) cast of a single snow flake, prepared without cover slip and examined at low magnification consists of marginal contours
and additional impressions which correspond with the regional thickness and three-dimensional profile of the crystal arms. In standard techniques, only the outlines are clearly visible, whereas the profile inside
the crystal arms can solely be perceived in the new techniques. Even the smallest roughness in the embedding material is visible. By adjusting the height of the condenser, the image changes
from brightfield-dominated (Fig. 21b) to darkfield-dominated (Fig. 21c).
Fig. 21: Snow flake, varnish cast preparation,
objective 4x, span length: 1.0 mm,
brightfield (a), condenser-based concentric brightfield- (b) and darkfield- (c) dominated VBDC, standard darkfield (d)
Also very fine superficial textures and reliefs in transparent crystallizations are visualized with superior clarity even at low magnification when concentric VBDC is carried out.
An example is given in
Fig. 22, showing a specimen of vitamin C. Only VBDC (Fig. 22c) reveals fine details that stay hidden in the brightfield image (Fig. 22a).
The contrast und three-dimensional appearance can be increased even further by colouring the light beams that generate the brightfield and darkfield images (Fig. 22d). Although the colouring may resemble the
corresponding polarisation image (Fig. 22b), some of the fine internal structures as well as their spatial arrangement in the object are perceptible with this bicolour contrast in higher clarity.
Fig. 22: Ascorbic acid, objective 4x,
HFW: 1.2 mm, brightfield (a), polarized light with quarter lambda compensator (b), concentric condenser-based VBDC in unfiltered halogen light (c) and bicolor light filtering (d)
Images of a diatom shown in Fig. 23 provide impressive evidence that VBDC increases the vertical resolution without affecting the lateral
resolution and that it also can be used advantageously at the maximum magnification range of the light microscope. Unsatisfactory results are obtained not only with a regular brightfield (Fig. 23a),
but also with a normal phase contrast (Fig.23b) and darkfield (Fig. 23d). As soon as VBDC is used (Fig. 23c
), blooming is reduced, the depth of field is many times greater and the fine lamellae dominate the foreground with their superior clarity.
Fig. 23: Small diatom (Cocconeis sp.) with linear pattern, HFW: 0.025 mm, thickness: circa 8 ĩm, objectice 100x, ocular 12.5x, brightfield (a),
phase contrast (b), condenser-based concentric VBDC (c), standard darkfield (d)
A Navicula alga (Fig. 24) can be used to demonstrate that phase-contrast-like views can also be obtained with normal brightfield objectives (without a phase ring!) when VBDC is used (see Fig. 24c). The new method reveals the fine lamellae of the diatom as well as its edge contours in best clarity and maximum depth of field.
Low density diatom (Navicula sp.), length: 0.05 mm, objective 100x, ocular 10x, brightfield (a), phase contrast (b), condenser-based eccentric VBDC (c)
Also in light stop-based VBDC, complex three-dimensional structures can be well visualized.
Continuous transitions between brightfield-
and darkfield-dominated views can be achieved by covering the illuminating light beam to varying degrees with the light stop. This is demonstrated with an alum crystal (Fig. 25).
This specimen was taken in low magnification, and VBDC was carried out with a light stop according to Fig. 3 (see principles
of VPDC). A moderate degree of coverage gives phase-contrast-like results (Fig. 25b and c); however, as soon as the light beam is completely covered, an axial (central) darkfield image results (Fig. 25d). Even a low degree of coverage (Fig. 25b)
accentuates fine contours that are barely revealed or are non-existent in brightfield (Fig. 25a). Also in this technical variant, the three-dimensional architecture is better accentuated in VBDC (Fig. 25b and c) and axial darkfield (Fig. 25d), when compared with standard bright- or darkfield imaging (Fig. 25a and e).
Fig. 25: Alum crystallization, objective 4x, HFW: 2.0 mm, brightfield (a), axial light stop-based VBDC, relative dominance of
brightfield (b) and darkfield (c), light stop-based axial darkfield (d), standard darkfield (e)
Also in higher magnification, the perception of three-dimensionality can be significantly improved with the help of light-stop based VBDC. As demonstrated in Fig. 26
, the architecture of the pyramidal alum crystallization taken with a 10 fold magnifying objective can only be seen in VBDC (Fig. 26c and d), whereas this pyramid appears as a flat polygon when observed in standard bright- or darkfield illumination (Fig. 26a and b).
Fig. 26: Pyramidal alum crystallization,
objective 10x, HFW: 0.5 mm, standard brightfield (a) and darkfield (b), light stop-based VBDC (c) and axial darkfield (d)
VBDC and axial darkfield are also well suited for software-based deep-focus stacking as the depth of field is higher in these methods than in other traditional techniques. In the Figures 27a
(color and B&W), foraminiferida are photographed in standard darkfield illumination, and the Figures 27b
(color and B&W) show the same specimens in axial darkfield achieved with the light stop shown in Fig. 3
according to the light pathway from Fig. 4a. To create the deep focus view shown in the the Figures 27b, a sequence of 7 single images was taken in different
focal planes and superimposed by stacking software in a second step. Combine Z
(Hedley, 2011), Picolay (Cypionka, 2011) and
Helicon Focus (Heliconsoft, 2011) are well tested for this task. In normal darkfield, multiple fine foramina cannot be well seen in these
shells, because they are not hit by the illuminating light coming from the condenserīs periphery and running in oblique direction to the
specimen. The same foramina are clearly visible in axial darkfield, because they are perpendicularly illuminated. These
Figures demonstrate the high quality also achievable in B&W when VBDC is carried out.
Fig. 27: Foraminiferida (center:
Boliviona sp), HFW: 0.6 mm, objective 10x, standard darkfield (a), light stop based axial darkfield, deep-focus stack (b), illumination with monochromatic green light (λ = 500 nm), color images
(left), conversions in B&W (right)
VBDC or axial darkfield and traditional darkfield can sometimes lead to complementary visual information. In this case, two single images
taken in the one and the other technique can be digitally superimposed on each other so that the resulting final image presents all visual
information from both single images. An example for this sandwich technique is given in Fig. 28. The foraminiferidum shown here was first taken in standard darkfield (Fig. 28a) amd axial darkfield illumination (Fig. 28b). Whereas bright illumination of the internal supporting elements (ribbed trabecula) of the skeleton shown dominates the image in darkfield (Fig. 28a),
any small pores present cannot be adequately captured. Although these do appear in VBDC along with a greater depth of field (Fig. 28b), the internal supporting elements
are not brightly illuminated in spite of the greater degree of detail, which limits the imaging effect. When both images are superimposed, a
darkfield-like image is obtained in which the pores and supporting elements are brightly contrasted (Fig. 28c).
Fig. 28: Foraminiferidum (Cibicidoides
sp), HFW: 0.3 mm, objective 10x, standard darkfield (a), light stop-based axial darkfield (b), software-based sandwich from images a and b (c)
Last Update: August 10th, 2012
Copyright: Timm Piper, 2012