All technical evaluations were made with Leitz/Leica laboratory microscopes. The condenser´s numerical aperture NA was 0.9 (dry system). VBDC could be carried out with objectives ranging from 2.5x up to
100x magnification. For VPDC and VPBC, 10-40 fold magnifying plane
achromatic and apochromatic phase contrast lenses (dry systems)
were used; the objective´s maximum numerical aperture was 0.75. Photomicrographs were taken
with a 7.1 MP digital camera (Olympus Camedia C-7070) and a 5 MP system camera from The Imaging Source (DFK 72AUC02), each mounted with Leitz/Leica photo-oculars. The white balance was automatically managed by the
cameras used (preset: indoor shooting, artificial light) so that the colorization of digital photomicrographs was comparable with that of the corresponding microscope images observed by the eye. Thus, all colors
visible in the photomicrographs presented were also apparent in the life images
observed. Depending on the particular properties of the specimen shown, the colors visible in the figures presented here are either real natural
colors or artificial colors generated by bicolor light filtering, or caused by interferences in appropriately thin specimens.
The microscopes used are shown in Figures 17 and 18. VBDC was
carried out with a microscope Leitz/Leica “Dialux” equipped with a Zernike Universal condenser (Fig. 17), VPDC and
VPBC were achieved with a Leitz/Leica HM Lux 3 microscope (Fig. 18) fitted with
several slides which could be slit into the cendenser. The cameras used for photomicrographs or video clips are demonstrated in the Figures 19 and 20. The Olympus digital camera (Fig. 19) could be
mounted with a Leitz/Leica vario photo ocular (range of magnification: 5-12.5x) when used with the trinocular photo tube of the Dialux microscope. The system camera from The Imaging Source (Fig. 20) could be
adapted with a Leitz/.Leica photo ocular Periplan GF 10x so that it could be put into the connecting pieces of each monocular, binocular or trinocular tube instead of a normal eyepiece. Alternatively, the Olympus
camera could also be connected with a Periplan GF-ocular as could the system camera with the vario ocular, so that both types of cameras could be used with both microscopes.
Microscope Leitz/Leica Dialux
Fig. 18: Microscope Leitz HM Lux 3
Fig. 19: Mounting of the Olympus digital camera with the trinocular phototube of the Dialux microscope
1 = vertical connecting piece for the vario photo ocular
2 = Leitz/Leica vario photo ocular 5-12.5x
3 = connecting cable for a flashlight
4 = ring-shaped adapter for connecting the vario photo ocular (manufactured by Promicron Comp. Germany)
5 = Olympus adapter-tube for connecting the digital camera
6 = cable of the Olympus AC adapter for power supply
Fig. 20: System camera DFK 72AUC02 mounted with a Leitz/Leica photo ocular Periplan GF
The new methods developed were tested with several unstained transparent specimens (varnish-casts of snow flakes, crystallizations of alum and
ascorbic acid, frustules of radiolarians and diatoms, living algae, wings of insects, permanent slides from marine Polychaeta). Just one
figure showing foraminiferida illuminated in axial darkfield resulted from deep-focus stacking. Apart from this particular image, no
stacking software for creating digital deep-focus or high dynamic range (HDR) reconstructions was used for preparing our
photomicrographs so that single shot images are presented showing the respective specimens in the same manner as visualized in the corresponding live observations.
Material for VBDC:
In order to achieve concentric VBDC, several universal condensers for phase contrast equipped with a high number of different
sized light annuli were combined with selected objectives; in suitable objectives, the diameter of the objective´s cross section area
was larger than the inner diameter of the corresponding condenser annulus and smaller than its external diameter.
Moreover, the numerical aperture of the objective
was lower than the condenser´s aperture. In this way, a set of
differently magnifying objectives could be selected and assigned to properly sized light annuli.
The other variants of VBDC (condenser-based eccentric and light stop-based VBDC) could be carried out with any type of
objective as long as the universal condenser was equipped with a large-sized light
annulus. As prototypes, two different types of light stops were available: A light stop designed as twin diaphragm as shown in fig. 3
(see section “principles of VBDC”) and a modified light stop fitted with a single polarizer inserted in one of
the two holes of the twin diaphragm.
Material for VPDC:
Several differently sized light annuli were made as prototypes for VPDC and combined with
different strength phase contrast
lenses. The sizes of the internal light annuli corresponding
with the respective phase contrast image were adapted to those of the
phase rings within the objectives used so that phase annuli and phase rings were conjugate. The diameters of the external darkfield
producing light annuli were adjusted with the objective´s internal cross section area so that these annuli were projected outside the
objective´s lenses. Light annuli of this design
were mounted on slides as presented in Fig. 8 (see section “principles of VPDC”) and
slit into the filter holder of a standard condenser so that VPDC could be achieved in transmitted light by use of a normal light microscope equipped with conventional phase contrast lenses.
As prototypes for axial VPDC, several slides carrying a light annulus for phase contrast were also fitted with an additional central perforation for axial darkfield illumination; examples are given in Fig. 10
(see section “principles of VPDC"). Phase contrast objectives containing normal phase rings were additionally fitted with a discoid light stop in centered position, i.e. in the middle of the phase ring. Fig. 12
shows a hand-made cylindrical light stopping objective insert designed for a phase contrast lens from Leitz /
Leica being well suited for this task. The alignment of this light stop and an appropriate condenser light mask is demonstrated in Fig. 13
(see section “principles of VPDC”). In the examples shown here, the breadth of the light annulus and the weighting of the phase
contrast and axial darkfield partial images can be regulated with the aperture diaphragm.
In the light masks used, centric perforation and light annulus could be filtered at different colors so that additional contrast effects were achievable based on colorized light.
Material for VPBC:
Also for VPBC, several differently sized light annuli were made as prototypes and combined with differently magnifying phase
contrast lenses. As described above, the sizes of the light annuli for phase contrast corresponded with those of the phase rings within
the objectives used, so that light annuli and phase rings were conjugate. In the first type of light masks for VPDC, a perforation was
made in the middle of the light annulus provided for phase contrast so that an axial bright-field image could be added. Other light
masks were fitted with peripheral perforations which could be used for concentric peripheral bright-field illumination. The
diameters of these external bright-field producing perforations were adjusted with the objective´s internal cross section area so that
they were projected in the periphery of the objective´s lenses. In
the prototypes used, the bright-field producing light was filtered in red,
and the complementary light associated with phase contrast was filtered in blue. So-designed light masks were mounted on slides as presented in Fig. 15
(see section “principles of VPBC”) and slit into the filter holder of a standard condenser so that VPDC
could be achieved in transmitted light by use of a normal light microscope equipped with conventional phase contrast lenses. The
alignment of light masks and phase ring could be controlled with a phase telescope as shown in Fig. 16 (see section “principles of VPBC”).
August 10th, 2012
Copyright: Timm Piper, 2012