The most important parameter in the basic 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.
The tutorial initializes with an illustration of the optical components of a phase contrast microscope displaying light waves passing from the illumination source (Lamp) to the intermediate image plane (Image Plane). In order to operate the tutorial, use the Phase Plate slider to toggle between brightfield illumination and positive or negative phase contrast. As the phase plate is altered by the slider, the relationship between the surround, diffracted, and resultant particle waves is illustrated. The Optical Component Opacity slider can be employed to reduce the opacity of the components to aid visualization of the waves passing through the virtual microscope.
The amplitude of the surround (undeviated) light must be reduced and the phase advanced or retarded (by a quarter wavelength) in order to maximize differences in intensity between the specimen and background in the image plane. The mechanism for generating relative phase retardation is a two-step process, with the diffracted waves being retarded in phase by a quarter wavelength at the specimen, while the surround waves are advanced (or retarded) in phase by a phase plate positioned in or very near the objective rear focal plane. Only two specialized accessories are required to convert a brightfield microscope for phase contrast observation. A specially designed annular diaphragm, which is matched in diameter and becomes optically conjugate to an internal phase plate residing in the objective rear focal plane, is placed in the condenser front focal plane.
The condenser annulus (illustrated in Figure 1) is typically constructed as an opaque flat-black (light absorbing) plate with a transparent annular ring, which is positioned in the front focal plane (aperture) of the condenser so the specimen can be illuminated by defocused, parallel light wavefronts emanating from the ring. The microscope condenser images the annular diaphragm at infinity, while the objective produces an image at the rear focal plane (where a conjugate phase plate is positioned, as discussed below). It should be noted that many texts describe the illumination emergent from the condenser of a phase contrast microscope as a hollow cone of light with a dark center. This concept is useful for describing the configuration, but is not strictly accurate. The condenser annulus either replaces or resides close to the adjustable iris diaphragm in the front aperture of the condenser. When conducting phase contrast experiments utilizing a condenser with both a phase annulus and an aperture diaphragm, check to ensure that the iris diaphragm is opened wider than the periphery of the phase annulus. Unlike differential interference contrast and Hoffman modulation contrast, the circular geometry of phase contrast illumination and detection enables specimen observation without orientation-dependent artifacts. Phase contrast is also insensitive to polarization and birefringence effects, which is a major advantage when observing tissue culture cells growing in plastic vessels.
Under conditions of Köhler illumination, surround light waves that do not interact with the specimen are focused as a bright ring in the rear focal plane of the objective (the diffraction plane). Under these conditions, the objective rear focal plane is conjugate to the condenser front aperture plane, so non-diffracted (zeroth order) light waves form a bright image of the condenser annulus at the rear aperture of the objective (superimposed over the image of the phase plate). The spherical wavefront that is diffracted by the specimen (the D waves) passes through the diffraction plane at various locations across the entire objective rear aperture, with the distribution (amount and location) depending on the number, size, and refractive index differential of light-scattering objects in the specimen. In contrast, the surround planar wavefront traverses a smaller region of the objective, which corresponds to the conjugate of the condenser annulus. The two wavefronts do not overlap to a significant extent, and occupy distinct portions of the objective rear focal plane. Because the direct, zeroth order light and diffracted light become spatially separated in the diffraction plane, the phase of either wave component (surround, S or diffracted, D) can be selectively manipulated without interfering with the other.
It is important to note that the diffraction pattern formed at the objective rear focal plane is the Fourier transform of all spatial frequencies deviated and scattered by the specimen in phase contrast and all other forms of optical microscopy. Consequently, the image produced at the intermediate image plane and the final image observed through the eyepieces (or recorded by a detector) represent inverse Fourier transforms of the diffraction patterns formed at the objective rear focal plane and the eyepoint (floating above the eyepiece front lens), respectively. Phase contrast microscopy takes advantage of these optical conjugate properties to enhance image contrast by modifying the microscope aperture function to introduce spatial filtration of specific image information. Introduction of a phase plate (filter) in the objective rear diffraction plane enables transformation of specimen phase variations into intensity variations that can be observed in the final image.
In order to selectively alter the phase and amplitude of the surround (or undeviated) light passing through the specimen, a phase plate is mounted in or near the objective rear focal plane (see Figure 1). In some phase contrast objectives, the thin phase plate contains a ring etched into the glass that has reduced thickness in order to differentially advance the phase of the surround (S) wave by a quarter-wavelength. Often, the ring is also coated with a partially absorbing metallic film to reduce the surround light amplitude by 70-90 percent. Because the objective rear focal plane often resides inside a lens element, other phase contrast objectives are produced by etching the surface of a lens that is closest to the diffraction plane. Regardless of how the objective is manufactured, the most important point to remember is that all phase contrast objectives are modified to include phase rings, a feature that is absent from all other microscope objectives.
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.