To achieve phase contrast, two components of bright field microscopes have to be modified as follows:
1. The condenser has to be equipped with a ring-shaped aperture or mask (the condenser annulus), which is placed near the condenser aperture diaphragm.
2. A conjugate phase plate (or ring) is placed in the back focal plane of the objective.
The condenser annulus and the phase-ring in the objective have to be optically aligned so that they are conjugate.
The correct alignment of the condenser annulus and the corresponding phase ring within the objective can be controlled by a phase telescope.
With this arrangement the specimen is concentrically illuminated by the apex of a cone of light. The light beams which are diffracted by the specimen pass the objective lens at various angles which are dependent on
the relative refractive index and the thickness of the specimen. The other light components, corresponding to the background, pass through the phase ring in the objective which produces an additional phase
difference. Thus, the phase differences between the specimen, its details and the background are amplified in the final image, so that minimal differences in refractive index are visible even in colourless specimens
with a low contrast and thickness.
Depending on the configuration and properties of the phase ring in the objective, the phase contrast microscopy can be positive or negative. In positive phase contrast the specimen is visible with medium or dark grey
features, surrounded by a bright halo; the background is of higher intensity than the specimen. In negative phase contrast the background is darker and the specimen appears brighter, surrounded by a dark halo.
The bright and dark halos are artifacts which are one of the major disadvantages of phase contrast; they are especially prevalent in specimens inducing large phase shifts.
The technical characteristics of phase contrast are described by several authors (1, 2, 3, 4, 9, 10).
Recently, advances in the design of objective phase-ring configurations have led to a new technique which reduces halo-effects callec apodized phase contrast (1, 3). Apart from the fact that the haloing is
reduced, the images of apodized phase contrast do not differ from conventional phase contrast images.
The figure demonstrates the light path of usual phase contrast and the final alignment of the condenser anulus and the phase ring.
Simplified optical pathway for phase contrast microscopy
(modified from 5)
Alignment of condenser annulus (bright) and phase ring (dark)
1 = light source
2 = annular shaped light mask (condenser annulus)
3 = condenser
4 = specimen
5 = background light
6 = light bent by the specimen
7 = phase ring
8 = eyepiece with intermediate image
9 = eye
Copyright: Joerg Piper, Bad Bertrich, Germany, 2007