Examine the light pathways through a phase contrast microscope and learn how these systems dissect the incident electromagnetic wave into a surround (S), diffracted (D), and resultant particle (P) component. The net result is that regions with very high optical path differences begin to appear bright. Home » Microscopy » Phase contrast Microscopy- definition, principle, parts, uses, Last Updated on February 24, 2020 by Sagar Aryal. Phase-contrast microscopy is basically a specially designed light microscope with all the basic parts in addition to which an annular phase plate and annular diaphragm are fitted. These effects are further accentuated by optical path difference fluctuations, which can turn bright halos dark in positive phase contrast, and dark halos bright in negative phase contrast. This module allows insertion of a phase annulus at a plane conjugate to the objective back aperture. An overview of positive phase contrast is presented in the upper portion of Figure 6. If the amplitudes of the particle and surround waves are significantly different in the intermediate image plane, then the specimen acquires a considerable amount of contrast and is easily visualized in the microscope eyepieces. Because the phase plate is positioned in or very near the objective rear focal plane (the diffraction plane) all light passing through the microscope must travel through this component. The slight phase shift of 1/20th wavelength exhibited by the resultant particle wave (which arises from interference between the diffracted and surround waves) is typically observed for minute details in a cell, and is related to the optical path length difference. A dark halo surrounds the specimen image when viewed with negative phase contrast optics (Figure 8(e)), and the shade-off transition ranges from bright at the edges to darker gray levels in the center. Explore the effects of changes to refractive index and thickness on optical path length, and discover how two specimens can have different combinations of these variables but still display the same path length. Cells that appear inconspicuous and transparent in brightfield can be viewed in high contrast and rich detail using a phase contrast microscope. The appearance of shade-off effects in relatively flat planar specimen areas, along with the excessively high contrast produced by edges and boundaries, provides strong evidence that the phase contrast mechanism is primarily controlled by the combined phenomena of diffraction and scattering. Apodized phase plates are useful for reducing the severity of halo, and specialized variable phase contrast systems can be fine-tuned to control these effects in order to optimize image quality and the fidelity of information obtained by the technique. Phase contrast does not require cells to be killed, fixed or stained. In addition, phase contrast microscopy is widely employed in diagnosis of tumor cells and the growth, dynamics, and behavior of a wide variety of living cells in culture. This thick or thin area in the phase plate is called the conjugate area. In fact, quantitative refractometry is often utilized to obtain refractive index values and for identification purposes. The conjugate area contains the material responsible for altering the phase of the surround (undiffracted) light by either plus or minus 90-degrees with respect to that of the diffracted wavefronts. When thin specimens are examined using positive phase contrast optics, which is the traditional form produced by most manufacturers, they appear darker than the surrounding medium when the refractive index of the specimen exceeds that of the medium. In negative phase contrast, the objective phase plate contains an elevated ring that retards the phase (rather than advancing the phase as in positive phase contrast) of the zeroth-order surround wave by a quarter-wavelength relative to the phase of the diffracted wave. Halo and shade-off artifacts depend on both the geometrical and optical properties of the phase plate and the specimen being examined. PHASE CONTRAST MICROSCOPE ALIGNMENT ensure that all objectives contain phase plates and are firmly seated in the nosepiece. The objectives and annular plates should also be sequentially ordered in their arrangement from lower to higher magnification Although these patterns occur as a natural result of the phase contrast optical system, they are often referred to as phase artifacts or image distortions. 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