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 (usually in culture), microorganisms, thin tissue slices, lithographic patterns, fibers, latex dispersions, glass fragments, and subcellular particles (including nuclei and other organelles).

Presented in Figure 2 is a comparison of living cells in culture imaged in both brightfield and phase contrast illumination. The cells are human glial brain tissue grown in monolayer culture bathed with a nutrient medium containing amino acids, vitamins, mineral salts, and fetal calf serum. In brightfield illumination (Figure 2(a)), the cells appear semi-transparent with only highly refractive regions, such as the membrane, nucleus, and unattached cells (rounded or spherical), being visible. When observed using phase contrast optical accessories, the same field of view reveals significantly more structural detail (Figure 2(b)). Cellular attachments become discernable, as does much of the internal structure. In addition, the contrast range is dramatically improved.

Phase relationships between the surround, diffracted, and particle (S, D, and P) waves in the region of the specimen at the image plane for brightfield microscopy (in the absence of phase contrast optical accessories) are presented in Figure 3. The surround and particle waves, whose relative amplitudes determine the amount of specimen contrast, are illustrated as red and green lines (respectively). The wave produced by diffraction from the specimen, which is never directly observed, is depicted as a blue wave of lower amplitude. The surround and diffracted waves recombine through interference to generate the resultant particle wave in the image plane of the microscope. The amplitude of each wave illustrated in Figure 3 represents the sum of the electric vectors of the individual component waves.

The most important concept underlying the design of a phase contrast microscope is the segregation of surround and diffracted wavefronts emerging from the specimen, which are projected onto different locations in the objective rear focal plane (the diffraction plane at the objective rear aperture). In addition, 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 optically conjugate to an internal phase plate residing in the objective rear focal plane, is placed in the condenser front focal plane.

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 in 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 regions are collectively 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 plate 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.

The phase plate configurations, wave relationships, and vector diagrams associated with the generation of positive and negative phase contrast images are presented in Figure 6. In addition, examples of specimens imaged by these techniques are also illustrated. As previously discussed, the spherical wavefront of diffracted light emerging from the specimen plane is retarded by a quarter-wavelength relative to the phase of the planar surround (or undiffracted) wavefront. In the positive phase contrast optical configuration (upper row of images in Figure 6), 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.

Identification of the properties of individual objectives is usually very easy because important parameters are often inscribed on the outer housing (or barrel) of the objective itself as illustrated in Figure 1. This figure depicts a typical 60x plan apochromat objective, including common engravings that contain all of the specifications necessary to determine what the objective is designed for and the conditions necessary for proper use.