Specimen Contrast Enhancement with Apodized Phase Plates

Phase contrast microscopy takes advantage of minute refractive index differences within cellular components and between unstained cells and their surrounding aqueous medium to produce contrast in these and similar transparent specimens. Recent advances in objective phase plate configuration have resulted in a new technique termed apodized phase contrast, which allows structures of phase objects having large phase differences to be viewed and photographed with outstanding clarity and definition of detail.

The tutorial initializes with a randomly selected digital image, captured with a phase contrast microscope, appearing in the Specimen Image window. Beneath the viewport is a pull-down menu labeled Choose A Specimen, which can be used to select a new specimen from the palette. To the right of the viewport appears a pair of phase plates positioned at different viewing angles and having the retardation film surrounded by neutral density filters with a default transmission value of 25 percent. The Apodized Phase Ring Neutral Density slider controls film density (and transmission values) of the neutral density apodized phase rings. Moving the slider to the right increases apodized phase ring neutral density to a maximum of 50 percent, while translating the slider to the left decreases the density to a minimum (zero density or transparent; transmission equals 100 percent). As the slider is moved to the right and left, the image in the Specimen Image window changes to reflect the effect that apodized phase plate neutral density has on specimen appearance.

An unfortunate artifact in phase contrast microscopy is the halo effect, which results in spurious bright areas around phase objects or reverse contrast in images. This effect is especially prevalent with specimens that induce large phase shifts. Reducing the halo artifact was once thought to be a difficult theoretical problem, but can be accomplished by the utilization of selective amplitude filters located adjacent to the phase film in the phase plates built into the objective at the rear focal plane. These amplitude filters consist of neutral density thin films applied to the phase plate surrounding the phase film. The transmittance of the phase shift ring in the classical phase plate is approximately 25 percent, while the two adjacent rings surrounding the phase shift ring in the apodized plate have a neutral density with 50 percent transmittance. The width of the phase film in both plates is the same. These values are consistent with the transmittance values of phase shifting thin films applied to standard plates in phase contrast microscopes. Nikon manufactures several apodized phase contrast objectives.

Contributing Authors

Matthew Parry-Hill, Cynthia D. Kelly, and Michael W. Davidson - National High Magnetic Field Laboratory, 1800 East Paul Dirac Dr., The Florida State University, Tallahassee, Florida, 32310.

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Specimen Contrast Enhancement with Apodized Phase Plates