Principles of 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

In VPDC, the specimen is simultaneously illuminated by two  different components of transmitted light which lead to two partial images, one phase contrast and one darkfield image. The darkfield illumination can be generated with concentric-peripheral light as usual in a normal darkfield microscope or it can be carried out with axial light so that axial (central) darkfield is achieved. Both partial images (the phase contrast and the respective darkfield image) are optically superimposed and interfere with each other. The amplitude of both illuminating light components and thus also the intensity and brightness of the partial images can be separately regulated by the user. In this way, the character of the optical contrast can be modified in tiny steps, and the appearance of the resulting image can be continuously changed from a darkfield-dominated to a phase contrast-dominated image. Moreover, VPDC can be achieved based on concentric or eccentric (oblique) illumination. Parts of the illuminating light beams can be covered in any definite direction by a facultative light stop which can be integrated into the illuminating light path if oblique illumination is required. In order to achieve VPDC, the condenser has to be designed in a particular manner. In my new illumination technique, the condenser aperture diaphragm can be used for modulations of the image´s appearance.

By the optical means described above, low density phase structures and high density light reflecting components are simultaneously visualized within the specimen. The phase contrast partial image is based on the principal zeroth order maximum and achieved by phase shift of the transmitted light. In the darkfield partial image, the principal zeroth order maximum does not contribute to the resulting image, because the corresponding illuminating light beams run past the objective lens. For this reason, darkfield images are only based on secondary maxima and reflected and scattered light components (Determann and Lepusch, 1981b). The particular contrast effects of my new method result from the superimposition and interference of these completely different partial images. The three-dimensional appearance and plasticity of the specimen can be improved, because the two illuminating light components associated with phase contrast and darkfield illumination are different with regard to their angle of incidence. The background brightness and the brightness of irradiated structures can be modulated by optical means (condenser iris diaphragm, polarization techniques) so that ultra-high ranges in brightness and contrast are avoided.


Optical solutions for concentric-peripheral VPDC  

When VPDC is carried out based on concentric-peripheral darkfield, normal phase contrast objectives equipped with a phase plate and a phase ring are necessary. A brightfield condenser or a universal condenser for phase contrast illumination has to be fitted with modified light masks. These masks can be shifted into appropriately constructed standard condensers when mounted on slides or integrated into the revolving turret of a universal condenser for phase contrast microscopy. The modified light masks consist of a pair of concentric light annuli: one light annulus for phase contrast illumination which has to be conjugate with the phase ring within the objective and another larger sized light annulus for darkfield illumination. The inner diameter of the latter annulus has to be somewhat larger than the diameter of the corresponding objective´s internal cross section area so that it is projected outside the objective. In this way, the specimen is concentrically illuminated in a phase contrast-like manner by transmitted light beams which come from the internal light annulus and run through the phase ring of the objective lens; additionally, it is also circularly illuminated in a darkfield-like manner by concentric light beams coming from the external light annulus which run in oblique direction through the specimen without reaching the objective lenses. In both so-generated partial images, the specimen is illuminated by concentric light cones at different angles of incidence or different steepness. The corresponding light path is demonstrated in Fig. 7.





Fig. 7: Light pathway for concentric-periopheral VPDC

  1 = light source
  2 = modified light mask fitted with a couple of concentric light annuli
  3 = condenser lens
  4 = specimen
  5 = illuminating light for phase contrast
  6 = illuminating light for darkfield
  7 = imaging light associated with phase contrast and darkfield
  8 = objective lens
  9 = phase plate with phase ring
10 = eyepiece with intermediate image
11 = eye

Figure modified from a schematic light pathway of normal phase contrast available at:
www.nobelprize.org/education/physics/microscopes/phase/index.html

 



The total area of the internal light annulus corresponding with phase contrast has to be much smaller than that of the external darkfield producing annulus. Otherwise, the darkfield image will be superseded by the phase contrast image. To achieve a well balanced proportion of these different partial images, special light masks should be created fitted with a few small internal perforations instead of the circular phase contrast producing light annulus normally used. Thus, the intensity of the illuminating light associated with phase contrast is significantly reduced when compared with standard light annuli consisting of a circular gap. A hand-made prototype of a suitable light mask leading to well balanced partial images in darkfield and phase contrast is shown in Fig. 8a, the correct alignment of this light mask and the phase ring of a compatible phase contrast objective controlled with a phase telescope is demonstrated in Fig. 8b. In VPDC, the internal light annulus is projected into the phase ring, whereas the external light annulus is not visible as situated outside the territory of the objective. The breadth of the darkfield-producing external light annulus can be regulated by the condenser aperture diaphragm. When this diaphragm is wide open, the final image will be dominated by the darkfield component, and phase contrast illumination will be dominant when the broadness of the external light annulus is decreased.





 

Fig. 8: Hand-made prototype of a condenser light mask for VPDC,
mounted on a slide (a), correct alignment of the internal perforations
and the objective´s phase ring, image taken with a phase telescope (b)


 

 

When the breadth of the external light annulus corresponding with the darkfield-like image is moderately reduced by the aperture diaphragm in tiny steps, potential darkfield-associated blooming and scattering can be attenuated, the background can be moderately brightened, ultra-high ranges in brightness and contrast can be equalized and the vertical resolution (focal depth) can be enhanced. When phase contrast objectives are designed as prototypes for advanced VPDC and fitted with an additional iris diaphragm mounted in their back focal plane, the objective´s aperture can be regulated in the same manner as well known in standard darkfield microscopy so that potentially remaining blooming and scattering can be eliminated still more and the depth of field can be further enhanced.

The projection of the condenser annuli and the run of the illuminating light components can be modified and adjusted in tiny steps when the condenser is moderately shifted in vertical direction - as well known in normal darkfied microscopy. Alternatively, the condenser´s head lens group could be designed as a zoom lens system so that its focal intercept could be continuously changed.

To obtain oblique illumination, a light stop can be shifted or integrated into the condenser as mentioned above so that one or both light annuli are partially covered, or the circular light annuli shown in Fig. 8 could be replaced by segmental arched narrow perforations.


Optical solutions for axial VPDC  

The light path in axial VPDC achieved with a modified phase contrast microscope is schematically shown in Fig. 9a, a detail view of a modified phase plate fitted with central light stop and phase ring is schematically shown in Fig. 9b together with the corresponding illuminating light beams. In axial VPDC, the specimen is illuminated first by a small axial light beam which is congruent with the optical axis. This beam is covered within the objective by a small light stop situated in the middle of the objective’s cross section area, i.e. in the centre of the phase ring, near or in the back focal plane. The specimen is illuminated further by a concentric light cone which passes the phase ring mounted within the objective according to the common technical standard. In this optical arrangement, two different partial images are superimposed: an axial darkfield image and a phase contrast image.

Fig. 9: Light pathway of APDC (a) and phase plate modified for APDC (b) . 1 = light source, 2 = modified light mask,
2.1. = light annulus for phase contrast, 2.2. = centric perforation for axial darkfield, 3 = illuminating light for phase contrast (3.1.) and axial darkfield (3.2), 4 = condenser lens, 5 = specimen, 6 = objective lens, 7 = modified phase plate,
7.1 = phase ring, 7.2 = centric light stop, 8 = imaging light for phase contrast and darkfield (8.1 and 8.2),
9 = eyepiece with intermediate image, 10 = eye


The condenser’s light mask has also to be modified and fitted with an additional small central perforation situated in the middle of the respective light annulus necessary for phase contrast. This central perforation has to be congruent with the objective’s light stop. Examples of such light masks mounted on slides which can be shifted into the condenser are presented in Fig. 10. In normal circumstances, the intensity of illuminating light needed for adequate image brightness is much higher in darkfield illumination than in phase contrast examinations. Thus, the phase contrast and darkfield partial images have both to be equalized in axial VPDC with regard to their brightness. To achieve this, the breadth of the phase contrast producing condenser annulus can be minimized so that this light ring is very narrow (Fig. 10a ) or this light annulus can consist of small perforations instead of transparent light segments (Fig. 10b). When the breadth of the light annulus is somewhat greater according to the size in normal phase contrast (Fig. 10c) it can also be reduced by closing the condenser aperture diaphragm.





Fig. 10: Condenser slides for APDC fitted with modified light masks, light annulus in reduced breadth (a),
replaced by annular perforations (b), made in normal size (c), slides a and b designed for 10x and 16x, slide c for 25x and 40x magnifying objectives, labelling from Fig. 9
 







The alignment of the condenser’s light mask (central and annular perforations) and the objective’s light stopper and phase ring can be controlled by a phase telescope. Some typical constellations visible with a phase telescope are schematically shown in Fig. 11. The central light stopper is surrounded by the phase ring (Fig. 11a ); the condenser light annulus is projected into the phase ring, and the additional small central light outlet situated within the optical axis is completely covered by the light stop (Fig. 11b). The breadth of the condenser’s light annulus and thus the intensity of the phase contrast image can be modulated by the aperture diaphragm. When the phase contrast producing annulus is completely covered by the maximum closed aperture diaphragm, the specimen is only illuminated by the remaining central axial light so that it appears in axial (central) darkfield illumination. When the aperture diaphragm is moderately opened, the transparent area of the condenser annulus is enhanced in tiny steps so that a phase contrast image is added and superimposed with the axial darkfield image. The intensity of the phase contrast partial image can be continuously regulated so that the appearance of the resulting VPDC-image can be changed from a darkfield-dominated to a phase contrast-dominated image in tiny steps.





Fig. 11: Light modulating elements for APDC,
controlled with a phase telescope,
brightfield illumination (a),
 correct alignment for APDC (b),
labelling from Fig. 9




In order to achieve variable phase contrast based on axial (central) darkfield, phase contrast objectives containing normal phase rings have additionally to be fitted with a discoid light stop in centered position (in the middle of the phase ring). Fig. 12 shows a hand-made cylindrical light stopping objective insert designed for a 10x phase contrast lens. The alignment of this light stop and an appropriate condenser light mask is demonstrated in Fig. 13 . 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.









Fig. 12: Hand-made prototype of a modified objective for APDC,
Leitz Phaco 10/0.25 (left), cylindrical insert with a small centric light stopper (right)




 









Fig. 13: Alignment of the prototype from Fig. 12 and a conjugate  light mask modified for APDC, brightfield (a), APDC (b-d), aperture diaphragm wide open, dominance of phase contrast (b),moderately closed, equalized illumination or relative dominance  of darkfield (c), maximum closed, only axial darkfield remaining  (d)





In order to achieve oblique illumination in any definite direction, parts of the illuminating light beams can be covered by a facultative light stop which has to be integrated into the illuminating light path.



Adding of brightfield illumination

In both variants of VPDC, the concentric-peripheral and the axial mode, a low intensity brightfield image can be added as third partial image on demand so that the specimen is illuminated in a “triplex mode”.

It can be achieved, when the maximum external diameter of the condenser light annulus is somewhat greater than that of the phase ring. Alternatively, the condenser light mask used has to be slightly turned into a moderate off-centered position so that a small part of the illuminating light transmitted through the specimen runs beside the phase ring within the objective. On this way, a brightfield-like partial image is added and superimposed with the darkfield and the remaining phase contrast image. The more the condenser annulus is 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.



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