Further developments in VPDC

Copyright: Jugend forscht

Timm Piper´s
microcopy site
Introduction
Principles of VBDC
Principles of VPDC
Principles of VPBC
Materials and Methods
Results of VBDC
Results of VPDC
Results of VPBC
Further developments
in VBDC
Further developments
in VPDC
Further devepolments
in VPBC
Optical calculations
Discussion
Summarizing remarks
and conclusions
Supplementary
images
Video Downloads
References
List of Personal 
Publications
Patents
Acknowledgements
About me
Contact Details

Technical developments in concentric-peripheral VPDC

In concentric-peripheral VPDC, the intensity of the phase contrast- and darkfield-like partial images can also be regulated by polarization techniques. For this purpose, at least the internal condenser light annulus, which represents the phase contrast partial image, can be fitted with a polarizer. In addition to this, a separate rotatable polarizer can be mounted beneath the condenser (Fig. 49a). In this arrangement, the intensity of the phase contrast producing light can be continuously regulated by turning the rotatable polarizer (see bottom arrow in Fig. 49a), whereas the intensity of the superimposed darkfield image can be adjusted with the condenser aperture diaphragm (not drawn in Fig. 49) by narrowing the external light annulus.



Fig. 49: Construction planes for condenser light masks fitted with analyzers and combined with a rotatable polarizer
mounted beneath the analyzers, internal zone covered by an analyzer (a), internal and external zones with two separate analyzers in crossed position (b). 1 = rotatable polarizer, 2 = analyzer for filtering the internal light annulus, 3 = light mask with internal and external annuli, 4 = rotatable light mask containing a modified external light annulus fitted with an annular analyzer. Further explanations in the text.


When the internal and external light annuli are each fitted with two concentric polarizers in crossed position, the light amplitudes of both zones can be regulated by the additionally inserted rotatable polarizer in an antagonistic manner without turning the aperture diaphragm or varying the intensity of the light source (Fig. 49b). The intensities of both illuminating light components can be regulated still more in higher variance if at least one of the concentric polarizers, preferably the external ring polarizer, is mounted as a rotatable light filter (see upper arrow in Fig. 49b).

Concentric-peripheral VPDC can also be integrated in incident light microscopy according to the construction plan shown in Fig. 50 (technical drawing based on and modified from E. Leitz Wetzar, 1969b). In order to achieve this, a vertical illuminator has to be altered so that the peripheral illuminating light beams contribute to a darkfield image based on epi-illumination, whereas an additional small incident annular illuminating light cone produces an epi-phase contrast image.



Fig. 50: Vertical illuminator modified for VPDC in incident light, darkfield (1) and phase contrast (2)
producing light, reflected imaging light (3), light mask with internal and external light annuli (4),
phase plate with phase ring (5).


As demonstrated in Fig. 50, a slide fitted with a pair of two appropriately sized light annuli could be shifted into the illuminator so that the illuminating light is separated into two parts leading to a phase contrast and a darkfield image. Both illuminating light components have also to be separated within the objective which can be designed in the same manner as usual in wafer microscopy but has additionally to be fitted with an appropriate phase ring. In this arrangement, the darkfield-associated illuminating light runs through the peripheral zone of the objective, and it is separated from the imaging light. The other illuminating light component which produces the phase contrast image runs through the imaging objective lenses and has to be conjugate with the objective´s phase ring. Both partial images, the phase contrast and darkfield image are superimposed in the plane of the intermediate image. Also in this technique, oblique illumination could be obtained in incident light when the illuminating light beams are partially covered within the vertical illuminator. The intensities of both different illuminating light components could be separately regulated when appropriately shaped polarization filters were integrated into the vertical illuminator and combined with a second rotatable polarizer– according to the technical principles which have been already described for transmitted light. Moreover, an iris diaphragm could be inserted into the vertical illuminator so that the breadth of the external illuminating light zone associated with the darkfield image could be reduced on demand by closing this diaphragm in tiny steps. Last, a small additional iris diaphragm could also be integrated into the objective´s imaging lens system so that the quality of the epi -darkfield image and the depth of focus could be enhanced further in the same manner as usual in transmitted light examinations.

In all variants of VPDC carried out with transmitted or incident light, additional bicolor double contrast effects can be achieved, when both illuminating light components associated with the phase contrast- and darkfield-like partial image are filtered in different, preferably complementary colors. Monochromatic light filters can therefroe be used for further optimization of resolution, sharpness and contrast if neceessary, especially in the case that images have to be converted in black and white.

When the internal light annulus which corresponds with phase contrast is slightly turned into a moderate off-centered position, a small part of the illuminating light transmitted through the specimen runs beside the phase ring within the objective so that a brightfield-like partial image is added and superimposed with the darkfield and the remaining phase contrast image. The more condenser annulus and phase ring are misaligned the higher are intensity and dominance of the brightfield partial image and the less the remaining phase contrast partial image can contribute to the resulting final image.



Technical developments in axial VPDC

Also in axial VPDC, manufacturers could create special condensers based on polarization techniques suitable for regulation of the intensity in the partial images generated so that the aperture diaphragm is no longer needed for regulating the dominance of phase contrast and axial darkfield. Suggestions for construction planes are shown in Fig. 51. The condenser light annulus, which represents the phase contrast partial image, can be fitted with an annular polarizer and the additional centric perforation with a discoid polarization filter. These filters should both preferably be mounted as rotatable components. In addition to this, a separate rotatable polarizer must be inserted into the illuminating light path beneath the so-modified light mask. In this arrangement, the intensities of all illuminating light components can be continuously regulated independent from each other by turning the rotatable polarization filters (Fig. 51a). When the centric light perforation and the phase contrast producing light annulus are each fitted with two concentric polarizers in fixed crossed position, the light amplitudes of both of these  zones can be regulated by the additionally inserted rotatable polarizer in an antagonistic manner without the light mask’s polarizers needing to be pivoted (Fig. 51b).



Fig. 51: Construction planes of condenser light masks for APDC fitted with polarization filters,
centric perforation and light annulus covered by rotatable (a) or fixed and crossed discoid (P1)
or annular (P2) polarization filters (b), additional rotatable polarizer (P3) beneath the light mask.
2 = modified light mask, 2.1. = light annulus for phase contrast, 2.2. = centric perforation for axial darkfield


Instead of polarizers, variable double diaphragms could also be integrated into special light masks for axial VPDC; some suggestions for construction plans are presented in Fig. 52. Thus, a small iris diaphragm could be mounted in centred position so that the diameter of the central perforation could be continuously regulated (Fig. 52a ); in the example shown here, the size of the external light annulus is fixed so that the breadth of this annulus could be regulated with the aperture diaphragm, if the condenser is fitted with one. Alternatively, the central perforation could be fixed, whereas the external light annulus could be regulated by an iris diaphragm integrated into the light mask (Fig. 52b). As shown in Fig. 52c , central perforation and light annulus could also both be fitted with iris diaphragms so that they could be regulated independently from each other.



Fig. 52: Suggestions for condenser light masks fitted with double diaphragms,
central iris diaphragm and fixed light annulus (a), fixed central perforation and
variable light annulus (b), double iris diaphragm system (c)


Manufacturers could also create variant objectives for paraxial phase darkfield contrast. Within such lenses, the centric light stopper could be replaced by a non transparent annular light stop mounted concentrically with the objective’s phase ring; the condenser light mask could be designed as a double ring system consisting of a couple of concentric light annuli for phase contrast and paraxial darkfield (light pathway in Fig. 53 ). In this variant, all axial imaging light components can pass the objective and contribute to the final image, but the specimen will no longer be perpendicularly illuminated by axial light. It would depend on the particular properties of the specimen, which variant might lead to superior results.







Fig. 53: Light pathway for paraxial phase-darkfield-contrast.

  1 = light source,
  2 = modified light mask,
  2.1. = light annulus for phase contrast,
  2.3. = light annulus for paraxial darkfield,
  3 = illuminating light for phase contrast (3.1.) and paraxial darkfield (3.3),
  4 = condenser lens,
  5 = specimen,
  6 = objective lens,
  7 = modified phase plate, 7.1 = phase ring, 7.3 = annular light stopper,
  8 = imaging light for phase contrast and darkfield (8.1 and 8.2),
  9 = eyepiece with intermediate image,
10 = eye









Moreover, special objectives for axial VPDC could be created fitted with miniaturized transparent plane parallel slides which could be pushed into the objective’s shaft near the phase plate or removed on demand. Such slides could act as carriers for discoid or annular light stoppers as schematically shown in Fig. 54 ; corresponding condenser light masks have to be conjugate with these light stoppers and the objective’s phase ring. Thus, axial or paraxial phase-darkfield-contrast and normal phase contrast could easily be carried out using the same objective by changing or removing the respective objective’s slide.

Fig. 54: Transparent slides for shifting into special phase contrast lenses for variable axial (a)
or paraxial (b) phase-darkfield contrast. 1 = discoid light stop, 1a = annular light stop


In principle, mirror lenses may be predestined for axial VPDC because the backside of their centric convex mirror acts as a light stopper in normal circumstances so that the axial light components cannot pass such objectives. While a centric light stop might be regarded as “foreign body” in any glass lens, it is a standard component in each mirror lens and absolutely necessary for use. In order to achieve axial VPDC with a mirror lens, an appropriately designed phase ring had to be inserted in concentric position near the axially mounted convex mirror. Of course, the condenser must be fitted with a well matching light mask so that the axial illuminating light is completely blocked by the backside of the centric mirror and the illuminating light cone which corresponds with phase contrast is congruent with the objective’s phase ring. Thus, some technical advantages of mirror lenses such as long working distances, loss of chromatical aberration and full correction also in ultraviolet and infrared light were also apparent in axial VPDC. A schematic light path is presented in Fig. 55.



Fig. 55: Catoptric mirror objective, fitted with a phase ring, designed for APDC (construction plan modified from Suemmchen, 2011)

 lm = light mask, ss = specimen slide, cs = cover slip,
1  = centric convex mirror,
2  = concave principal mirror,
3  =  illuminating light for axial darkfield,
3a = illuminating light for phase contrast,
4  = imaging light,
5  = centric perforation,
5a = light annulus


 

 





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