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.
The tutorial initializes with a randomly selected specimen appearing in the Phase Contrast Image window, and the corresponding wave relationships giving rise to the phase image being displayed in the graph to the immediate left of the image window. In order to operate the tutorial, use the mouse cursor to translate the Phase Contrast Mode slider between Positive and Negative phase contrast or Brightfield illumination. As the slider is translated, the image appearing in the Phase Contrast Image window changes to display how the specimen appears in the current imaging mode set by the slider. In addition, beneath the waveform graph is a Phase Plate that changes shape to conform with the imaging mode selected by the slider. In order to view a new specimen, utilize the Choose A Specimen pull-down menu to select another example.
The phase plate configurations, wave relationships, and vector diagrams associated with the generation of positive and negative phase contrast images are presented in Figure 1. In addition, examples of specimens imaged by these techniques are also illustrated. In the positive phase contrast optical configuration (upper row of images in Figure 1), the surround (S) wavefront is advanced in phase by a quarter-wavelength when traversing the phase plate, to produce a net phase shift of 180 degrees (one half wavelength). The advanced surround wavefront is now able to participate in destructive interference with the diffracted (D) waves at the intermediate image plane. In most cases, merely advancing the relative phase of the surround wavefront alone is insufficient to result in the generation of high-contrast images in the microscope. This occurs because the amplitude of the surround waves is significantly larger than that of the diffracted waves and suppresses the resulting images created by interference from only a small portion of the total number of waves. In order to reduce the amplitude of the surround wavefront to a value closer to that of the diffracted wave (and enforce interference at the image plane), the phase ring in the objective is increased in opacity by application of a semi-transparent metallic (neutral density) coating. Surround light waves, which pass almost exclusively through the phase ring by design in the phase contrast microscope, are dramatically decreased in amplitude by the opacity of the phase plate to a value that ranges between 10 and 30 percent of the original intensity.
Because the resultant particle wave is produced exclusively by interference of the surround and diffracted wavefronts, interference between the wavefronts arriving at the image plane generates a particle (P) wave with an amplitude that is now considerably less than that of the surround when the neutral density coating is applied. The net effect is to transform the relative phase difference introduced by the specimen into a difference in amplitude (intensity) of the light emerging from the image plane. Because the human eye interprets differences in intensity as contrast, the specimen is now visible in the microscope eyepieces, and can also be captured using a digital camera system. All positive phase contrast systems selectively advance the phase of the linear surround (S) wavefront relative to that of the spherical diffracted (D) wavefront. Specimens having a higher refractive index than the surrounding medium appear dark on a neutral gray background, while those specimens that have a lower refractive index than the bathing medium appear brighter than the gray background.
In order to modify the phase and amplitude of the spatially separated surround and diffracted wavefronts in phase contrast optical systems, a number of phase plate configurations have been introduced. Because the phase plate is positioned at or very near the objective rear focal plane (the diffraction plane) all light passing through the microscope must travel through this component. The portion of the phase plate upon which the condenser annulus is focused is termed the conjugate area, while the remaining areas are referred to as the complementary area. The conjugate area contains the material responsible for altering the phase of the surround (undiffracted) light by either plus or minus 90-degrees with respect to that of the diffracted wavefronts. In general, the phase ring conjugate area is wider (by about 25-percent) than the region defined by the image of the condenser annulus in order to minimize the amount of surround light that spreads into the complementary area.
Most of the phase plates available from modern microscope manufacturers are produced by vacuum deposition of thin dielectric and metallic films onto a glass plate or directly onto one of the lens surfaces within the microscope objective. The role of the dielectric thin film is to shift the phase of light, while the metallic film attenuates undiffracted light intensity. Some manufacturers utilize multiple anti-reflective coatings in combination with the thin films to reduce the amount of glare and stray light reflected back into the optical system. If the phase plate is not formed on the surface of a lens, it is usually cemented between successive lenses that reside near the objective rear focal plane. The thickness and refractive indices of the dielectric, metallic, and anti-reflective films, as well as those of the optical cement, are carefully selected to produce the necessary phase shift between the complementary and conjugate areas of the phase plate. In optical terminology, phase plates that alter the phase of surround light relative to diffracted light by 90 degrees (either positive or negative) are termed quarter wavelength plates because of their effect on the optical path difference.
An overview of positive phase contrast is presented in the upper portion of Figure 1. Positive phase contrast plates (left-hand side of Figure 1) advance the surround wave by a quarter-wavelength due to the etched ring in the glass plate that reduces the physical path taken by the waves through the high refractive index plate. Because the diffracted specimen rays (D) are retarded by a quarter-wavelength when interacting with the specimen, the optical path difference between the surround and diffracted waves upon emergence from the phase plate is one-half wavelength. The net result is a 180-degree optical path difference between the surround and diffracted waves, which results in destructive interference for a high refractive index specimen at the image plane. The amplitude profiles of the destructively interfering waves for positive phase contrast are depicted in the upper graph of Figure 1. The amplitude of the resultant particle (P) wave is lower than the surround (S) wave, causing the object to appear relatively darker than the background, as illustrated by the image of Zygnema green algae at the far right (labeled POS). A vector diagram illustrating advancement of the surround wave by a quarter-wavelength, which is shown as a 90-degree counterclockwise rotation in positive phase contrast, appears between the graph and the image in Figure 1.
It is also possible to produce microscope optical systems yielding negative phase contrast, as illustrated in the lower portion of Figure 1. In this case, the surround (S) wave is retarded (rather than being advanced) by a quarter-wavelength relative to the diffracted (D) wave. The result is that specimens having high refractive indices appear bright against a darker gray background (see the image labeled NEG in the lower portion of Figure 1). In negative phase contrast, the objective phase plate contains an elevated ring that retards the phase (rather than advancing the phase as in positive phase contrast) of the zeroth-order surround wave by a quarter-wavelength relative to the phase of the diffracted wave. Because the diffracted wave has already been retarded by a quarter-wavelength when passing through the specimen, the optical path difference between the surround and diffracted waves is eliminated, and constructive interference occurs for a high refractive index specimen at the image plane. Note that the resulting particle (P) wave is higher in amplitude than the surround (S) wave in negative phase contrast (see the lower graph in Figure 1). Also illustrated is the vector diagram for negative phase contrast, where the surround wave vector undergoes a 90-degree clockwise rotation.
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.