Phase Contrast Microscopy

A large spectrum of living biological specimens are virtually transparent when observed in the optical microscope under brightfield illumination. To improve visibility and contrast in such specimens, microscopists often reduce the opening size of the substage condenser iris diaphragm, but this maneuver is accompanied by a serious loss of resolution and the introduction of diffraction artifacts. Phase contrast was introduced in the 1930's for testing of telescope mirrors, and was adapted by Zeiss laboratories into a commercial microscope several years later. This technique provides an excellent method of improving contrast in unstained biological specimens without significant loss in resolution, and is widely utilized to examine dynamic events in living cells.
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Phase Contrast Microscopy

Introduction to Phase Contrast

Research by Frits Zernike uncovered phase and amplitude differences between zeroth order and deviated light that can be altered to produce favorable conditions for interference and contrast enhancement.

Fluorescence and Phase Contrast Combination Microscopy

To minimize the effects of photobleaching, fluorescence microscopy can be combined with phase contrast illumination.

Phase Plate/Ring Alignment

Concentric alignment of the condenser phase plate slits with the phase ring, positioned inside the objective, is of paramount importance in phase contrast microscopy. This tutorial explores the effect of phase plate/ring alignment on specimen contrast using this important microscopy technique.

Employing both classical and apodized phase contrast techniques, The Evident Scientific Microscopy Resource Center phase contrast digital image gallery features a wide spectrum of specimens illuminated with this useful contrast enhancing technique. Among the specimens illustrated in the phase contrast gallery are fossilized bone thin sections, stained plant tissue sections, butterfly wing scales, cells in tissue culture, algae, protozoa, and histology specimens.

Phase Contrast References

A number of excellent books, review articles, and original research reports on phase contrast microscopy have been published by leading researchers in the field and were utilized as references to prepare the phase contrast discussions included in the MicroscopyU website. This section contains periodical location information about these articles, as well as providing a listing of selected original research reports and books describing specimen contrast and the classical techniques of phase contrast light microscopy.

Contributing Authors

Mortimer Abramowitz - Olympus America, Inc., Two Corporate Center Drive., Melville, New York, 11747.
Matthew Parry-Hill, Robert T. Sutter, Cynthia D. Kelly, Shannon H Neabes, Omar Alvarado and Michael W. Davidson - National High Magnetic Field Laboratory, 1800 East Paul Dirac Dr., The Florida State University, Tallahassee, Florida, 32310.