In conventional phase contrast, the condenser annulus is completely transparent, so that the passing ligth beams create a light cone which illuminates the specimen in a concentric circular manner (360 °). When the
condenser annulus and the objective phase ring are correctly aligned, both illuminating elements are exactly conjugate; the f alignment is usually controlled using a centering telescope (phase telescope).
To achieve relief-phase-contrast, the transparent annulus has to be replaced by a smaller transparent sector (sectoral light mask), which can be crescent-shaped or a small round aperture. This light sector has to be
aligned with the phase ring in the objective in the usual manner, so that it is conjugate. With this modification, the specimen is only illuminated from one defined direction by oblique light beams.
In most cases, the resulting images have more contrast, focal depth and three dimensionality and less visible spherical aberration compared with conventional phase contrast.
Crescentic or circular-shaped sectoral masks can be achieved in several ways, controlled using a phase telescope (see fig. 3):
1. Sliding components with an annular ring can be moved into the path of light and the condenser aperture diaphragm can be partially closed, so that just a small part of the annular ring remains transparent for
2. Condenser turrets with a kit of several annular rings, usually existing in Zernike phase condensers, can be used for relief-phase-contrast, if the turret is rotated into an abnormal position, so that
annular ring and phase ring overlap slightly. The condenser aperture diaphragm is closed as described above.
The individual position of the phase ring in the objectives is not important for realising relief phase contrast, because the position of the illuminating light sector can be adjusted to the phase ring with a high
degree of variability. Thus, objectives made by different manufacturers can be used simultanously.
Moreover, the condenser annuli can be directly modified as when they are covered by an opaque plate with a small aperture for the transmitted light. In this case it is not necessary to close the condenser aperture
diaphragm (see fig. 4). Alternatively, the annulus could also be replaced by other constructions, suitable to achieve circumscribed small light beams, which can be adjusted to the phase rings in
the objective (see. Discussion).
As in normal phase contrast, the quality of the resulting images can be optimized also in relief-phase-contrast by closing the field diaphragm so that it is just seen at the edges of the field of view (KÖHLER
The images demonstrate the light path and the alignment of the light modulating elements in relief phase contrast.
Simplified optical pathway
for relief phase contrast microscopy
Alignment of sectoral light mask (bright) and phase ring (dark)
1 = light source
2 = modified condenser light mask
3 = condenser
4 = specimen
5 = background light
6 = light bent by the specimen
7 = phase ring
8 = eyepiece with intermediate image
9 = eye
Implementation of relief phase contrast using non transparent slides and several ring-shaped apertures (images taken with a phase telescope):
(a) The condenser aperture diaphragm
is partially closed and the margin of the slide is moved in from the right (shadowed area of the aperture). The darker ring is the phase ring of the objective.
(b) Final alignment of the margin of the
slide and the aperture diaphragm.
(c) An annular ring for phase contrast (small diameter, bright) is shifted from the right, overlapping the phase-ring (dark).
(d) Final alignment with partially closed
aperture diaphragm, visible on the right (dark)
(e) An annular ring for dark field (large diameter, bright) is overlapping the phase ring (dark) on the left side. The aperture diaphragm (dark) is partially
closed, its edges visible on the left.
(f) Final alignment with partially closed aperture diaphragm.
Correct alignment of a special light mask for relief phase contrast (small sectoral transparent gap, bright) and the phase ring (dark). The condenser aperture diagpragm is open.
Image taken with a phase telescope.
A prototype of this light mask is shown in fig. 5b.
Copyright: Joerg Piper, Bad Bertrich, Germany, 2007