Differences in light absorption are often negligible between living cells and their surrounding nutrient medium, as well as between the various intracellular components and plasma membranes, rendering these entities barely visible when observed by brightfield illumination. 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.
A mechanism to translate variations in phase into corresponding changes in amplitude.
Phase contrast optical components can be added to virtually any brightfield microscope.
Using phase-related optical techniques to increase specimen contrast.
Advanced phase ring technology that enables a reduction in image halo artifacts.
Apodized Phase Contrast
Explore how specimen size affects the angle of diffracted light rays that pass through apodized phase plates.
Optical Pathways in the Phase Contrast Microscope
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.
Phase Contrast Microscope Alignment
Learn how to align a phase contrast microscope and examine variations in specimen appearance through the eyepieces (at different magnifications) when the condenser annulus is shifted into and out of alignment with the phase plate in the objective.
Positive and Negative Phase Contrast
This interactive tutorial explores relationships between the surround (S), diffracted (D), and resulting particle (P) waves in brightfield as well as positive and negative phase contrast microscopy.
Shade-Off and Halo Phase Contrast Artifacts
Explore shade-off and halo artifacts, where the observed intensity does not directly correspond to the optical path difference (refractive index and thickness values) between the specimen and the surrounding medium.
Specimen Optical Path Length Variations
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.
Selected Literature References
Contrast enhancement using interference of polarized light wavefronts.
A listing of pertinent books, review articles, and research reports on phase contrast.
Using refractive index variations to produce optical contrast in transparent specimens.
Examine the origins of contrast in a wide spectrum of specimens.
Douglas B. Murphy - Department of Cell Biology and Anatomy and Microscope Facility, Johns Hopkins University School of Medicine, 725 N. Wolfe Street, 107 WBSB, Baltimore, Maryland 21205.
Ron Oldfield - Department of Biological Sciences, Division of Environmental and Life Sciences, Macquarie University, New South Wales 2109, Australia.
Stanley Schwartz - Bioscience Department, Nikon Instruments, Inc., 1300 Walt Whitman Road, Melville, New York 11747.
Greenfield Sluder - Department of Cell Biology, University of Massachusetts Medical School, 377 Plantation Street, Worcester, Massachusetts 01605.
Tatsuro Otaki - Optical Design Department, Instruments Company, Nikon Corporation, 1-6-3 Nishi-Ohi, Shinagawa-ku, Tokyo, 140-8601, Japan.
Matthew Parry-Hill, Robert T. Sutter, Cynthia D. Kelly, Shannon H. Neaves, Omar Alvarado, and Michael W. Davidson - National High Magnetic Field Laboratory, 1800 East Paul Dirac Dr., The Florida State University, Tallahassee, Florida, 32310.