Microscopy Primer
Light and Color
Microscope Basics
Special Techniques
Digital Imaging
Confocal Microscopy
Live-Cell Imaging
Microscopy Museum
Virtual Microscopy
Web Resources
License Info
Image Use
Custom Photos
Site Info
Contact Us

The Galleries:

Photo Gallery
Silicon Zoo
Chip Shots
DNA Gallery
Amino Acids
Religion Collection
Cocktail Collection
Screen Savers
Win Wallpaper
Mac Wallpaper
Movie Gallery

Phase Contrast Microscopy
Interactive Java Tutorials

Specimen Contrast Enhancement
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.



Questions or comments? Send us an email.
© 1995-2019 by Michael W. Davidson and The Florida State University. All Rights Reserved. No images, graphics, software, scripts, or applets may be reproduced or used in any manner without permission from the copyright holders. Use of this website means you agree to all of the Legal Terms and Conditions set forth by the owners.
This website is maintained by our
Graphics & Web Programming Team
in collaboration with Optical Microscopy at the
National High Magnetic Field Laboratory.
Last modification: Friday, Jun 15, 2018 at 11:41 AM
Access Count Since November 10, 2000: 17754
For more information on microscope manufacturers,
use the buttons below to navigate to their websites: