Principles of 
 Phase Contrast
Principles of 
 Relief Phase Contrast
Material and  
Further technical 
Material and Methods

Relief phase contrast was developed on two LEITZ microscopes ”Dialux” and ”HM-Lux III”. These microscopes are both able to achieve positive phase contrast. The Dialux was equipped with a Zernike phase contrast universal condenser, the HM-Lux III with a kit of separate slides for phase contrast and dark field which can be shifted into an existing bright field condenser. In this way, the several varieties of technical realisiation described above could be evaluated.

The images of the phase ring and annular ring constellations (see fig. 1-4), controlled by a phase telescope, were taken using Canon Powershot A 620 and Casio Exilim EX-Z 110 cameras. The microscopical images were taken using Olympus Camedia C 7070 and Canon EOS 350D / 20D cameras operated with remote switches. When using the Olympus Camedia C 7070 camera also life-movies of relief-phase-contrast images could be taken in an appropriate quality.

When slides for phase contrast are used (see fig. 5a), the margin of the illuminating sector can be built by the edge of the slide and the aperture diaphragm or by the annular ring itself and the aperture diaphragm as shown in fig. 3 a and b. Annular rings for dark field microscopy (see fig. 5c) which are characterized by much higher diameters can also be used according to fig. 3 e and f.

Moreover, a slide with an annular ring can directly be prepared for relief phase contrast when it is covered by a opaque black mask; a prototype of this is shown in fig. 5b. In this case, the condenser aperture diaphragm has to be in wide open. The corresponding phase telescope image is shown in fig. 4.

The turret of a Zernike condensor or condenser slides for phase contrast (see fig. 5a) can be rotated or shifted into several positions to achieve marginal overlappings of the respective light annulus and the phase ring as shown in fig. 3 c and d. The condenser aperture diaphragm has to be used for additional limitations of the illuminating field. Annular rings with various diameters can be used, so that the configuration of the resulting illuminating field can be variable. The images in fig. 3 show some examples for this..

Similar effects can also be achieved when special slides for relief phase contrast are used as shown in fig. 5 d and e. The condenser aperture diaphragm has to be partially closed as described above. When the aperture diaphragm is in a wide position, bright field illumination will result from this instead of relief phase contrast.

A binocular viewing tube should preferably be used to align the illuminating elements. In this way, one eyepiece can be removed and the phase-telescope can be inserted in its place.Thus, the effects of manipulations can be controlled simultanously by the remaining eyepiece. When an optimal alignment is found, the centering telescope can be replaced by the second eyepiece, so that normal binocular examination can then be made.

Fig. 5:
Standard condenser sliders for conventional phase contrast (a), relief phase contrast with a special mask (b), and dark field (c).
Prototype condenser sliders for relief phase contrast with semicircular (d) and circular (e) shaped masks.

Copyright: Joerg Piper, Bad Bertrich, Germany, 2007


[Principles of Phase Contrast]
[Principles of Relief Phase Contrast]
[Material and Methods]
[Further technical developements]