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.
The tutorial initializes with a randomly selected specimen image appearing in the Phase Contrast Image window on the right-hand side of the tutorial. Each specimen utilized in this tutorial is relatively thick and displays contrast effects that are dependent upon the objective phase plate configuration. In order to operate the tutorial, use the Phase Contrast Mode slider to vary specimen contrast between that observed with a standard dark low (DL) and a more dense (higher neutral density) dark medium (DM) phase plate.
As the slider is translated, the phase plate annular ring (above the slider) darkens to indicate reduced transmission and retardation properties, and many specimen features undergo contrast variations. In some cases, contrast inversion occurs (usually manifested in dark areas appearing much brighter) in the specimens appearing in the Phase Contrast Image window. Simultaneously with specimen contrast variations produced by translating the slider, the wave amplitude graph, positioned directly above the phase plate, changes to demonstrate a reduction in amplitude of the surround wave (red curve) relative to the resultant (or particle, the green curve) wave. Diffracted light waves, which maintain constant amplitude regardless of the phase plate characteristics, are indicated by the blue curve.
Variations in the light transmission and retardation values of the objective phase plate can induce a wide range of effects, resulting in very low contrast (approaching brightfield illumination) to a pseudo-darkfield effect. For a majority of specimens, the best results fall somewhere in between the extremes, producing a smooth gradation of brightness levels for fluctuating values of refractive index and thickness throughout the specimen. In addition, thin specimens often respond in a different manner than thicker specimens when observed under identical retardation (or surround wave advancement) and phase plate neutral density (transmission attenuation) values.
The tutorial compares the effects of two common objective phase plate configurations. Phase plates having the dark low (DL) specification are manufactured with a retardation value of one quarter wavelength and an average level of neutral density, resulting in specimen contrast that falls into the middle of the range. DL objectives produce a dark image outline on a light gray background, and are the typical objectives utilized for all-purpose phase contrast observation. These objectives are designed to furnish the strongest dark contrast in specimens having a major difference in refractive index from that of the surrounding medium. The DL phase contrast objective is the most popular style for examination of cells and other semi-transparent living material and is especially suited for photomicrography and digital imaging. Nikon manufactures DL phase contrast achromat, plan achromat, and plan fluor objectives.
Alternatively, the dark medium (DM) phase plate configuration has a smaller surround wave retardation value (approximately one-eighth of a wavelength), but a much higher neutral density than the dark low plate. Dark medium phase plates produce a dark image outline on a medium gray background. These objectives are designed to be used to achieve high image contrast with specimens having small phase shifts or refractive index differences, such as fine fibers, flagella, cilia, granules, and very small particles. Usually restricted to higher magnification objectives having large numerical apertures (fluorites and apochromats), DM phase contrast performs well with very thin specimens, but often displays a reversal of contrast when thick specimens (or specimens with a very high refractive index) are imaged. Nikon manufactures DM phase contrast plan apochromat objectives.
The image intensity observed in a phase contrast microscope (or recorded either digitally or with traditional film) does not bear a simple linear relationship to the optical path difference of the specimen. This concept is illustrated in Figure 1, which displays relative specimen intensity as a function of phase change for several positive contrast phase plates having a fixed retardation value (90 degrees) and differing levels of neutral density. The red curve corresponds closely to the dark medium (DM) objective, while the yellow curve approximates the dark low (DL) objective. The blue curve presents a phase plate having no neutral density added, and mimics a brightfield objective at a phase change of 90 degrees (see Figure 1). Specimens falling into the gray region of Figure 1 appear darker than the background, and those in the white region appear lighter than the background.
Contrast is greater at smaller optical path differences with objectives having higher neutral density values (the red curve; DM objective), but thicker specimens undergo contrast inversion at much lower path differences (approximately 50 degrees retardation). As a consequence, image interpretation can be difficult with thick specimens when using dark medium phase contrast objectives. For example, fine cellular cilia that have a refractive index only slightly higher than the surrounding medium (approximately 15 degrees retardation) will appear dark gray. However, thicker features, such as the nucleus or larger organelles, having retardation values around 60 percent, experience contrast inversion and appear very bright on a dark background. When examined with a phase contrast objective having a neutral density of 25 percent (yellow curve), the same specimen has a nucleus that is darker than the cilia. These dramatic differences in contrast can lead to quite different interpretations of cellular morphology.
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.
Kenneth R. Spring - Scientific Consultant, Lusby, Maryland, 20657.
Matthew Parry-Hill, Cynthia D. Kelly, and Michael W. Davidson - National High Magnetic Field Laboratory, 1800 East Paul Dirac Dr., The Florida State University, Tallahassee, Florida, 32310.