Relief Phase Contrast is a new technique for phase contrast light microscopy developed by the autor which lead to an improved quality of microscopic images,
especially in transparent thin-layer preparations. This method has been applied for a patent and published in English (13) and German (14).
Phase contrast is a mode of light microscopy that is widely used for the examination of transparent and colourless specimens such as unstained living cells and micro-organisms
which typically have very low contrast. These phase objects do not absorb light, so that the amplitude of the light waves passing through them remains nearly constant. However, they do modify the phase
of transmitted light by around one quarter wavelengh (lambda/4) or less. Such differences in phase cannot be perceived by the eye or by a photography.
In 1934/35, the dutch physicist Frits Zernike developed phase contrast as a new illumination mode to convert phase differences into visible amplitude differences (11-12)
for which he was awarded the 1953 nobel price (5).
To achieve phase contrast, the condenser and the objectives have to be modified. The condenser has to be equipped with annular shaped masks and a conjugate phase ring has to
be placed in the objective.
All technical modifications used phase contrast yet, do not create three-dimensional images which could be compared with the three dimensional effects of interference contrast
microscopy. Compared with bright field, in phase contrast the depth of focus is smaller, because the condenser aperture irisdiaphragm has to be fully open. In phase contrast, the intensity of contrast is
dependent on the differences of refractive indices of the specimen and the surrounding medium, and the thickness and native contrast of the specimen.
The quality of phase contrast images is strongly determined by the quality of the lenses. Existing chromatic and spherical aberrations reduce the quality of the resulting
images more intensively than in brightfield microscopy. In phase contrast, the specimens are surounded by a bright or dark halo. These halos are artifacts which are one of the major disadvantages of
phase contrast; they are especially prevalent in specimens inducing large phase shifts.
Furthermore, phase contrast can only be achieved when the phase rings in the objectives and the the condenser annuli are specifically adjusted to each other. Normally, a
particular ring-shaped mask can be used for one or two special objectives within a well-defined range of magnification. For example, one condenser annulus can useded with objectives for 10x or 16x
magnification, a second mask for 25x and 40x, and a third mask for 100x magnifiing oil-immersion objectives. When objectives and condensers are used from different manufacturers, a misalignment can
result. Therefore, the kit of objectives and condenser should perferably be from the same manufacturer.
Recently, relief phase contrast has been developed as a new modification of phase contrast which can improve the quality of the conventional phase contrast images by higher
contrast, enlarged focal depth, higher shapness, reduced haloing and less-visible spherical aberration. This method can also be used when existing phase-contrast objectives are built by different
manufacturers. Moreover, in most cases this technique requires only one light- modulating element in the condenser, which is suitable for all existing phase-contrast objective.
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Copyright: Joerg Piper, Bad Bertrich, Germany, 2007ight: