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.
Brief Overview of Phase Contrast - The search was still on in the early part of the twentieth century to find a way of using both direct and diffracted light from all azimuths to yield good contrast images of unstained objects that do not absorb light. Research by Frits Zernike during this period uncovered phase and amplitude differences between zeroth order and deviated light that can be altered to produce favorable conditions for interference and contrast enhancement.
Phase Contrast Microscopy - Phase contrast microscopy, first described in 1934 by Dutch physicist Frits Zernike, is a contrast-enhancing optical technique that can be utilized to produce high-contrast images of transparent specimens such as living cells, microorganisms, thin tissue slices, lithographic patterns, and sub-cellular particles (such as nuclei and other organelles). In effect, the phase contrast technique employs an optical mechanism to translate minute variations in phase into corresponding changes in amplitude, which can be visualized as differences in image contrast. One of the major advantages of phase contrast microscopy is that living cells can be examined in their natural state without being killed, fixed, and stained. As a result, the dynamics of ongoing biological processes in live cells can be observed and recorded in high contrast with sharp clarity of minute specimen detail.
Specimen Contrast in Optical Microscopy - Light can interact with a specimen through a variety of mechanisms to generate image contrast. These include reflection from the surface, absorption, refraction, polarization, fluorescence, and diffraction. Contrast can also be increased by physical modification of the microscope optical components and illumination mode, as well as manipulation of the final image through photographic or digital electronic techniques. The discussion in this section highlights various interactions between the specimen and light, and reviews some of the optical microscopy techniques that have been developed to enhance specimen contrast.
Apodized Phase Contrast - 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 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.
Comparison of Phase Contrast and DIC Microscopy - Phase contrast and differential interference contrast (DIC) microscopy are complementary techniques capable of producing high contrast images of transparent biological phases that do not ordinarily affect the amplitude of visible light waves passing though the specimen. The most fundamental distinction between differential interference contrast and phase contrast microscopy is the optical basis upon which images are formed. Phase contrast yields image intensity values as a function of specimen optical path length magnitude, with very dense regions (those having large path lengths) appearing darker than the background. The situation is quite distinct for differential interference contrast, where optical path length gradients (in effect, the rate of change in the direction of wavefront shear) are primarily responsible for introducing contrast into specimen images.
Fluorescence and Phase Contrast Combination Microscopy - To minimize the effects of photobleaching, fluorescence microscopy can be combined with phase contrast illumination. The idea is to locate the specific area of interest in a specimen using the non-destructive contrast enhancing technique (phase) then, without relocating the specimen, switch the microscope to fluorescence mode.
Phase Contrast Microscope Configuration - Phase contrast optical components can be added to virtually any brightfield microscope, provided the specialized phase ring objectives conform to the tube length parameters, and the condenser will accept an annular phase ring of the correct size. The major manufacturers all provide phase contrast accessories for their research and teaching-level microscopes, both in upright and inverted (tissue culture) configurations. This section outlines the necessary equipment for observing specimens in phase contrast illumination and discusses basic steps in microscope alignment.
Frits Zernike (1888-1966) - Frits Zernike was a Dutch mathematician and physicist who discovered the phase contrast phenomenon and was awarded the Nobel Prize in 1953. It was Zernike's studies in optics that ultimately led to his prestigious award. He first received evidence of the phase contrast phenomenon in a study of diffraction gratings, when he was able to selectively detect transparent materials with different refractive indices. In 1938, Zernike built a microscope based on phase contrast illumination, but it initially received little attention. At the time, a lack of specimen contrast experienced with common microscopic techniques was one of the major concerns in optical microscopy.
Phase Contrast Interactive Java Tutorials
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.
Diffracted Light in Phase Contrast Microscopy - In all forms of optical microscopy, the specimen scatters light through processes that include diffraction, refraction, reflection, and absorption. Transparent specimens imaged by phase contrast techniques diffract light that is retarded by one-quarter wavelength (90 degrees) with respect to undiffracted (surround) incident illumination, whereas opaque specimens, such as diffraction gratings, diffract light that is 180-degrees (one-half wavelength) out of phase with the surround illumination. This interactive tutorial explores diffraction of light by a periodic grating in a phase contrast microscope.
Phase Contrast Microscope Alignment - Careful alignment of the phase contrast microscope is essential to produce the maximum contrast effect without introducing artifacts that degrade specimen appearance. This MicroscopyU interactive tutorial examines variations in how specimens appear through the eyepieces (at different magnifications) when the condenser annulus is shifted into and out of alignment with the phase plate in the objective.
Optical Pathways in the Phase Contrast Microscope - The most important parameter in the design of a phase contrast microscope is to isolate the surround and diffracted light waves emerging from the specimen so that they occupy different locations in the diffraction plane at the rear aperture of the objective. This interactive tutorial explores light pathways through a phase contrast microscope and dissects the incident electromagnetic wave into surround (S), diffracted (D), and resultant (particle; P) components.
Phase Plate Configuration Effects on Specimen Contrast - The transmission and retardation properties of surround (undiffracted) light passing through the phase plate annulus in phase contrast microscopy can significantly affect the overall specimen contrast observed in the microscope. This interactive tutorial explores contrast variations induced by altering phase plate absorption and retardation characteristics.
Positive and Negative Phase Contrast - Depending upon the configuration and properties of the phase ring positioned in the objective rear focal plane, specimens can be observed either in positive or 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. In addition, phase plate geometry and representative specimen images are also presented.
Specimen Optical Path Length Variations - Phase contrast microscopy interprets differences in specimen optical path length as fluctuations in light intensity, which are readily observed as variations in contrast through the microscope. This interactive tutorial explores the effects of refractive index and thickness changes on the apparent overall optical path length, and demonstrates how two specimens can have different combinations of these variables but still display the same path length.
Interaction of Light Waves with Phase Specimens - Upon encountering a phase specimen, an incident illumination wavefront is deformed according to the geometry, refractive index, and thickness of the specimen. This interactive tutorial examines the variety of deformations observed in wavefront shape as specimens having differing characteristics are illuminated with a planar beam of light.
Shade-Off and Halo Phase Contrast Artifacts - Two very common effects observed in phase contrast images are the characteristic shade-off and halo patterns in which the observed intensity does not directly correspond to the optical path difference (refractive index and thickness values) between the specimen and the surrounding medium. This interactive tutorial demonstrates shade-off artifacts in positive and negative phase contrast microscopy.
Apodized Phase Contrast - In apodized phase contrast microscopy, halo attenuation and an increase in specimen contrast can be obtained 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 filter thin films applied to the phase plate surrounding the phase film as illustrated in the tutorial window.
Apodized Phase Plates and Specimen Contrast - 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.
Comparison of Phase Contrast and DIC Microscopy - The most fundamental distinction between differential interference contrast (DIC) and phase contrast microscopy is the optical basis upon which images are formed by the complementary techniques. Specimens examined by these contrast-enhancing methods produce images that are often quite different in appearance and character when objectively compared. This MicroscopyU interactive tutorial explores many of the similarities and differences exhibited between images captured with phase contrast and DIC microscopy.
Optical Sectioning with Phase Contrast and DIC - One of the primary advantages of differential interference contrast (DIC) microscopy over phase contrast is the ability to utilize the instrument at full numerical aperture without suffering the masking effects of phase plates or condenser annuli, which severely restrict the size of the condenser and objective apertures. The major benefit is improved axial resolution, particular with respect to the ability of the DIC microscope to produce excellent high-resolution images at large aperture sizes. This interactive tutorial explores and compares optical sectioning of thick specimens with DIC and phase contrast, and reveals the benefits of unrestricted aperture effects on obtaining well-defined sections.
Phase Contrast Digital Image Galleries
Phase Contrast Image Gallery - Employing both classical and apodized phase contrast techniques, the Molecular Expressions 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.
Positive and Negative Phase Contrast - Transparent specimens often appear remarkably different when comparatively observed under positive and negative phase contrast illumination. In positive phase contrast, specimen intensity is manifested by relatively medium to dark gray features, surrounded by a bright halo, and superimposed on a lighter gray background. Alternatively, in negative phase contrast, the specimen often appears much brighter on a dark gray background and the accompanying halos are also dark (much darker than the background). This digital image gallery compares identical viewfields of a wide variety of specimens illuminated with both positive and negative phase contrast.
Phase Contrast and DIC Comparison Image Gallery - Phase contrast and differential interference contrast (DIC) should be considered as complementary (rather than competing) techniques, and employed together to fully investigate specimen optical properties, dynamics, and morphology. In many cases, each technique will reveal specific details about a particular specimen that is not apparent from observing images captured by other methods. The wide variety of images presented in this MicroscopyU gallery are derived from both thick and thin transparent specimens, as well as specimens that have inherent contrast originating from synthetic dyes (stains) or natural pigments.
Selected Literature References
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.
Mortimer Abramowitz - Olympus America Inc., Two Corporate Center Drive., Melville, New York, 11747.
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.
Kenneth R. Spring - Scientific Consultant, Lusby, Maryland, 20657.
Matthew J. 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.
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