The principle of phase contrast is applied when the objects we want to observe are transparent. The technique was discovered in 1934 by Fritz Zernike, a Dutch physicist, who applied it to microscopy.
When a contrast is applied to a transparent sample or ‘phase object’, such as a dye into a cell, the specimen is converted into an ‘amplitude object’ since it produces an impression in the amplitude wavefront of the electromagnetic (EM) field.
In phase objects, either the optical thickness, or the refractive index, or both, vary from point to point throughout its volume. However, as the human eye cannot detect variations in the phase of the EM field, such objects are therefore invisible. This is the reason why subjects such as cells and tissue sections have to be stained, ie. converted into amplitude objects. When dealing with stains however, they can be unsatisfactory because sometimes they have a negative impact on biological processes resulting in artifacts and ultimately cell death.
Diffraction occurs when an EM field of constant phase encounters an object, resulting in a distortion in either the amplitude, phase or shape of the field. Consider an EM field propagating through a phase object such as a cell. The cell causes a retardation in a region of the wave front. The emerging wavefront is no longer planar but an impression is made corresponding to the area retarded by the cell. In this case, the wavefront is said to be phase modulated.
To be continued….