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.
The tutorial initializes with a rectangular specimen appearing in semi-transparent blue color on the left-hand side of the window. Adjacent to the specimen is a simulated phase contrast viewfield (entitled Ideal Image until the Shade-Off slider is translated) depicting how the specimen would appear in the microscope under ideal artifact-free conditions. On the right-hand side of the window is a Line Intensity Scan graph taken across the center of the specimen along the x-axis. This scan illustrates the intensity profile of the specimen with respect to the background.
In order to operate the tutorial, use the mouse cursor to translate the Shade-Off Degreeslider to the right, which will increase the severity of this artifact. As the shade-off value is increased, the appearance of the specimen in the Shade-Off Image window is altered to reflect changes to the specimen image. Increasing the level of the shade-off and halo artifacts redistributes intensity in the center of the specimen and produces a surrounding halo. Simultaneously, the Line Intensity Scan profile is altered to correspond with changes to the specimen image. Specimen contrast can be reversed by toggling between Positive Phase Contrast and Negative Phase Contrast with the radio buttons.
Although shade-off and halo patterns occur as a natural result of the phase contrast optical system, they are often referred to as phase artifacts or image distortions. In all forms of positive phase contrast, bright phase halos usually surround the boundaries between large specimen features and the medium. Identical halos appear darker than the specimen with negative phase contrast optical systems. These effects are further accentuated by optical path difference fluctuations, which can turn bright halos dark in positive phase contrast, and dark halos bright in negative phase contrast.
Halos occur in phase contrast microscopy because the circular phase-retarding (and neutral density) ring located in the objective phase plate also transmits a small degree of diffracted light from the specimen (it is not restricted to passing surround waves alone). The problem is compounded by the fact that the width of the zeroth-order surround wavefront projected onto the phase plate by the condenser annulus is smaller than the actual width of the phase plate ring. The difference in width between the phase ring and surround wavefront is usually around 25 to 40 percent, but is necessary due to restrictions and requirements of the optical design. Because of the spatial location of the phase ring in the objective diffraction plane, only those wavefronts corresponding to low spatial frequencies diffracted by the specimen pass through the annulus of the phase plate. Thus, the diffracted specimen waves passing through the phase plate remain 90-degrees (a quarter-wavelength) out of phase relative to the zeroth-order (undeviated or surround) light. The resulting phase contrast halo artifact is due to attenuation of the low spatial frequency information diffracted by the specimen through a very shallow angle with respect to the zeroth-order surround wavefronts. In effect, the absence of destructive interference between low spatial frequency wavefronts diffracted by the specimen and undeviated light waves produces a localized contrast reversal (manifested by the halo) surrounding the specimen. In order to create a sharp edge in the image, all of the spatial frequencies diffracted by the specimen must be represented in the final image.
Phase contrast halos are especially prominent and noticeable around large, low spatial frequency objects such as nuclei, diatoms, and entire cells. Another contributing factor to the halo artifact is the redistribution of light energy at the image plane, from regions where it is destructive to regions where it is constructive. Large, high contrast halos can produce confusing images for specimens generating large optical path differences, such as erythrocytes, molds, protozoa, yeast cells, and bacteria. On the other hand, halo effects can often emphasize contrast differences between the specimen and its surrounding background and can increase the visibility of thin edges and border details in many specimens. This effect is particularly helpful in negative phase contrast, which produces a dark halo surrounding low frequency image detail. In many cases it is possible to reduce the degree of phase shift and diffraction, resulting in reduced halo sizes around the specimen. The easiest remedy for removing or attenuating the intensity of halos is to modify the refractive index of the observation medium with higher refractive index components, such as glycerol, mannitol, dextran, or serum albumin. In some cases, changing the refractive index of the medium can even produce a reversal in image contrast, turning dark specimen features bright without significantly disturbing the background intensity.
The halo effect can be significantly reduced by utilizing specially designed phase objectives that contain a small ring of neutral density material surrounding the phase ring material near the objective rear aperture. These objectives are termed apodized phase contrastobjectives, and enable structures of phase objects having large phase differences to be viewed and photographed with outstanding clarity and definition of detail. In most cases, subcellular features (such as nucleoli) can be clearly distinguished as having dark contrast with apodized objectives, but these same features have bright halos or are imaged as bright spots using conventional phase contrast optics. With the apodized optics, contrast is reversed due to the large amplitude of diffracted light relative to that of the direct light passing through the specimen.
In practice, halo reduction and an increase in specimen contrast with apodized optical systems can be achieved 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. The transmittance of the phase shift ring in the classical phase plate is approximately 25 percent, while the pair of adjacent rings surrounding the phase shift ring in the apodized plate have a neutral density with 50 percent transmittance. The width of the phase film in both plates is the same. These values are consistent with the transmittance values of phase shifting thin films applied to standard plates in phase contrast microscopes.
Shade-off is another very common optical artifact in phase contrast microscopy, and is often most easily observed in large, extended phase specimens. It would normally be expected that the image of a large phase specimen having a constant optical path length across the diameter would appear uniformly dark or light in the microscope. Unfortunately, the intensity of images produced by a phase contrast microscope does not always bear a simple linear relationship to the optical path difference produced by the specimen. Other factors, such as absorption at the phase plate and the amount of phase retardation or advancement, as well as the relative overlap size of the phase ring and condenser annulus also play a critical role. The intensity profile of a large, uniformly thick positive phase contrast specimen often gradually increases from the edges to the center, where the light intensity in the central regions can approach that of the surrounding medium (the reverse is true for negative phase specimens). This effect is termed shade-off, and is frequently observed when examining extended planar specimens, such as material slabs (glass or mica), replicas, flattened tissue culture cells, and large organelles.
The effects of halo and shade-off artifacts in both positive and negative phase contrast are presented in Figure 1 for a hypothetical extended phase specimen having rectangular geometry and a higher refractive index than the surrounding medium (Figure 1(a)). The intensity profile recorded across a central region of the specimen is illustrated in Figure 1(b). In positive phase contrast (Figure 1(c)), the specimen image exhibits a distinctively bright halo and demonstrates a dramatic shade-off effect, which is manifested by progressively increasing intensity when traversing from the edges to the central region of the specimen (see the intensity profile in Figure 1(d)). The halo and shade-off effects have reversed intensities in negative phase contrast (Figure 1(e) and 1(f)). A dark halo surrounds the specimen image when viewed with negative phase contrast optics (Figure 1(e)), and the shade-off transition ranges from bright at the edges to darker gray levels in the center. In addition, the intensity profile (Figure 1(f)) is reversed from that observed with positive phase contrast.
The shade-off phenomenon is also commonly termed the zone-of-action effect, because central zones having uniform thickness in the specimen diffract light differently than the highly refractile zones at edges and boundaries. In the central regions of a specimen, both the relative amount and the angles of diffracted light are dramatically reduced when compared to the edges. Because diffracted wavefronts originating from the central specimen areas have only a marginal spatial deviation from the zeroth-order non-deviated surround wavefronts (but are still retarded in phase by a quarter-wavelength), they are captured by the phase plate in the objective rear focal plane, along with the surround light. As a result, the intensity of the central specimen region remains essentially identical to that of the background. The appearance of shade-off effects in relatively flat planar specimen areas, along with the excessively high contrast produced by edges and boundaries provides strong evidence that the phase contrast mechanism is primarily controlled by the combined phenomena of diffraction and scattering.
Halo and shade-off artifacts depend on both the geometrical and optical properties of the phase plate and the specimen being examined. In particular, the width and transmittance of the phase ring play a critical role in controlling these effects (the phase ring width is typically about one-tenth the total aperture area of the objective). Wider phase rings having reduced transmittance tend to produce higher intensity halos and shade-off, whereas the ring diameter has a smaller influence on the effects. For a particular phase objective (either positive or negative), the optical path difference and specimen size, shape, and structure have significant influence on the severity of halo and shade-off effects. In addition, these effects are heavily influenced by the objective magnification, with lower magnifications producing better images.
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.
Kenneth R. Spring - Scientific Consultant, Lusby, Maryland, 20657.
Matthew Parry-Hill and Michael W. Davidson - National High Magnetic Field Laboratory, 1800 East Paul Dirac Dr., The Florida State University, Tallahassee, Florida, 32310.