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.