Phase objects cause a phase shift of light passing through a specimen. Because only amplitude shifts (differences in intensities) are visible for the human eye or photo detectors, the staining of a specimen would mediate an amplitude shift and a difference in the intensity of light passing through. Many staining reagents are toxic for living cells, however. Phase contrast microscopy offers a possibility to use phase shifts caused by differences in optical path length to make a specimen visible under the optical microscope. It changes the phase shifts into amplitude shifts via the interference of resulting light waves.
The technique of phase contrast microscopy was developed in the 1930s by the Dutch physicist Frits Zernike. After the technique was brought into use in 1942, Zernike was awarded the Nobel Prize in Physics in 1953.
The optical path length is the product of the refractive index and the thickness between two points in an optical path. It is related to the transit time and the velocity of light. The differences in the optical path length lead to different velocities of the light waves when they pass through the specimen (i.e. phase shift). As a result differences in phase occur. A higher refractive index, compared to the surrounding medium, leads to a deceleration of the light wave and a retardation of its phase.
Interference describes the interaction of two waves with each other and the resulting formation of a new wave pattern following the principle of superposition. The relevant parameter for the interference of light waves is the light wave’s amplitude. If two waves interfere, the amplitude of the resulting light wave will be equal to the vector sum of the amplitudes of the two interfering waves.
If the amplitude of the resulting wave is increased, the interference will be described as constructive. This will be the case if either two wave crests or two wave troughs meet at the same point in time. It is also possible that a crest of one wave and a trough of another wave meet at the same point in time. This leads to a decreased amplitude of the resulting wave. The interference between these two waves is then called destructive.
The key elements of a phase contrast microscope are an annulus aperture and a phase plate. The annulus aperture is placed in the front focal plane of the condenser and limits the angle of the penetrating light waves. The phase plate lies in the back focal plane of the objective and has a phase ring made of a material that dims the light passing through it and changes its phase by λ/4. λ represents the light’s wavelength.
In phase contrast microscopy under the conditions of Köhler illumination the light waves which are not interacting with the specimen are focused as a bright ring in the back focal plane of the objective. The light ring spatially matches the phase ring along the optical axis and causes a phase shift of the undeviated light. Light that is diffracted by the specimen does not predominantly strike the phase ring and is therefore not affected.
Between the affected and the unaffected light waves there is a total phase shift of up to λ/2. The phase of undeviated light is advanced by λ/4 at the phase ring and diffracted light waves are usually retarded by λ/4 by biological specimens. The total phase shift of λ/2 allows destructive interference of the light waves in the image plane. To dim the undeviated light passing the phase ring, it is important to avoid an outshining of the undeviated compared to the deviated light.
A phase shift of λ/2, as observed in phase contrast microscopy, gives rise to a maximal destructive interference effect as crest and trough effectively meet at the same point in time. Therefore the amplitude of the light wave is reduced and the phase shift of the phase object is transformed into an amplitude shift.
There are two forms of phase contrast: positive and negative phase contrast. They mainly differ by the phase plates used for illumination. In positive phase contrast, the phase of light passing through the phase ring is advanced compared to the deviated light, whereas it is retarded in phase in negative phase contrast. The retardation of phase in negative phase contrast leads to a destruction of the phase differences. The light waves are in phase and instead of destructive interference, constructive interference occurs. This leads to an increased amplitude of the resulting light wave.
In positive phase contrast microscopy, objects with a higher refractive index than the surrounding medium are displayed darker than objects with a lower refractive index. For negative phase contrast the opposite applies.
Phase contrast microscopy visualizes differences in the optical path length of a specimen. The optical path length is related to the specimen’s thickness and the refractive index. Cellular structures like plasma membranes and organelles have a profound impact on the optical path length. As many cells (especially in cell cultures) have a flat and regular shape, they are hardly visible in brightfield microscopy.
A phase contrast image of such cells amplifies differences in cell structure and can be regarded as an optical density map, as optical density has a great influence on the refractive index of a specimen or material. However, several effects complicate the correct interpretation of the phase contrast image as they do not directly rely on differences in optical path length.
The halo effect describes the appearance of a bright edge for positive phase contrast or a dark edge for negative phase contrast around large objects. Halos form because some of the diffracted light from the specimen traverses the phase ring as well. The ring of light formed by the undeviated waves is a little bit smaller than the phase ring and low-spatial-frequency diffracted light waves from the specimen can pass through the annulus. The deviated light passing through the phase ring maintains a phase difference of 90° and is therefore not affected by destructive interference. This leads to a reversion in contrast and causes the halo at the boundaries of large objects.
The shade-off effect describes a situation where homogenous parts of a specimen are displayed with the same light intensity as the surrounding medium. Although the light passing through these regions experiences a phase shift, only minor diffraction occurs and the angle of scattering is greatly reduced. Therefore these light waves enter the phase ring like undeviated light and do not experience interference.
Another problem in phase contrast microscopy can be contrast inversion. If there are objects with a very high refractive index next to objects with a low refractive index, they will appear brighter instead of darker (for positive phase contrast). In such regions the phase shift is not the usual shift of λ/4 for biological specimens, and instead of destructive interference, constructive interference occurs (opposite for negative phase contrast).
Although these effects can render interpretation of phase contrast images difficult, phase contrast microscopy is a convenient and important optical contrast technique for imaging phase objects. Additionally, phase contrast microscopy enables the investigation of cellular functions and structures in live specimens, making it the most frequently applied contrast method in biological research.