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Phase Contrast Microscopy
Interactive Java Tutorials
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 ring 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 photomicrograph of a starfish embryo appearing in the virtual microscope viewport. 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 density to a maximum of 50 percent and moving the slider to the left decreases the density to a minimum of zero density (transparent; transmission equals 100 percent). As the slider is moved to the right and left, the image in the microscope viewport changes to reflect the effect that apodized phase plate neutral density has on specimen appearance.
The purpose of the neutral density film is to retard the phase of direct light passing through the specimen by one quarter wavelength to allow constructive and destructive interference with diffracted light at the intermediate image plane. On the right in the tutorial window is an illustration of an apodized phase plate positioned at two different angles. Surrounding the phase film in these plates are two concentric areas of semi-transparent neutral density material, which reduce the intensity of diffracted light from the specimen.
As the slider is moved to the left, note the halo surrounding the outer periphery of the starfish embryo and the lack of contrast and image detail present in the central portion of the cell mass. Significantly improved contrast is observed when the slider is moved to the right, increasing apodized phase plate neutral density and reducing the amount of light transmitted through the plate. When the slider is moved to the far right position, the starfish embryo has a dramatically reduced halo around the periphery and exhibits sharper edges with enhanced internal specimen detail. Similar effects are seen in other specimens available in the pull-down menu.
Contributing Authors
Mortimer Abramowitz - Olympus America, Inc., Two Corporate Center Drive., Melville, New York, 11747.
Matthew J. Parry-Hill 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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