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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 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. 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 microscopy. 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,
Details from native epithelial cells, higher corrected planapochromatic objective 40x, flash,
Piper, J.: Piper, J.: Relief-phase-contrast - a new technique for phase-contrast microscopy - Copyright: Joerg Piper, Bad Bertrich, Germany, 2010 |
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