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). Such phase differences cannot be perceived by the eye or by
In 1934/35, the dutch physicist Frits Zernike developed phase contrast as a new illumination mode to convert phase differences into visible amplitude differences.
Phase contrast examinations can be carried out as positive or negative phase contrast. In positive phase contrast the specimen is visible with medium or dark grey features, surrounded by a bright halo; the background
is of a 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.
Recently, advances in the design of objectives for phase contrast have led to a new technique which reduces halo-effects called apodized phase contrast microscopy.
All these modifications used in phase contrast do not create three-dimensional images which could be compared with 3D effects of interference contrast microscopy. Compared with brightfield, in phase contrast the
depth of focus is smaller, because the condenser aperture diaphragm is 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
Moreover, phase contrast can only be achieved when the phase rings in the objectives and the condenser annuli are specifically adjusted for each other. Therefore, a misalignment can result when objectives and
condensers are used from different manufacturers.
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, reduced haloing and
less-visible spherical aberration. In most cases, specimens occur in an enhanced sharpness and three dimensionality, similar to interference contrast images. The planarity of the microscopical image is
improved, especially when objectives are used which are not highly corrected. Even when highly corrected objectives are used (e.g. planachromatic or planapochromatic lenses), the images resulting from relief phase
contrast have more contrast, enlarged focal depth and more apparent three-dimensional aspects. Compared with interference contrast, relief phase contrast often produces images with higher or complementary
information of specimen details.
In contrast with conventional phase contrast, the
condenser aperture iris diaphragm can be moderately closed, as usual in brightfield microscopy. Thus, in relief phase contrast the image quality can be influenced by
this diaphragm similar to brightfield images
Relief phase contrast can also be achieved without any misalignments when existing phase contrast objectives from different manufacturers are used simultaneously.
To achieve this, the path of the illuminating light has to be modified. All details about technical aspects are described in a separate contribution (www.relief-phase-contrast.com).
Native epithelial cells, basic corrected achromatic objective 40x, white light,
conventional phase contrast (left), relief phase contrast (right)
Details from native epithelial cells, higher corrected planapochromatic objective 40x, flash,
relief phase contrast (left), interference contrast (right)
Relief phase contrast - an universally applicable modifikation of phase contrast with improved image quality (in German)
(submitted: 24 May 2006, accepted: 30 May 2006)
Mikrokosmos 96 / 4, 223-229, 2007
Piper, J.: Relief-phase-contrast - a new technique for phase-contrast microscopy -
(submitted: 19 June 2006, accepted: 23 June 2006)
European Microscopy and Analysis 21 / 4, 9-12, 2007
Copyright: Joerg Piper, Bad Bertrich, Germany, 2010