Fundamentals of Differential Interference Contrast Microscopy

Here is an article about the fundamentals of differential interference microscopy, and its configuration by the Olympus Microscopy Resource Center.

In the mid-1950s, a French optics theoretician named Georges Nomarski modified the Wollaston prism used for detecting optical gradients in specimens and converting them into intensity differences. Several implementations of this design, which is called differential interference contrast

Differential interference contrast microscopy, also known as Nomarski Interference Contrast (NIC) or Nomarski microscopy, is an optical microscopy illumination technique used to enhance the contrast in unstained samples. It works on the principle of interferometry to gain information about the optical density of the sample, and to see invisible characteristics. It uses a complex lighting system that produces an image with the object appearing black to white on a grey background, similar to phase contrast microscopy, but without the bright diffraction halo. It is a method of deriving contrast in an unstained specimen from differences in index of refraction of specimen components.

The DIFFERENTIAL INTERFERENCE CONTRAST MICROSCOPY works by separating a polarized light source into two beams which take slightly different paths through the sample. These will take different optical path lengths like product of refractive index and geometric path length. The emissions are then recombined at the image plane, where wave interference may occur. Image contrast can be modified by altering the phase difference between the reference and specimen rays. It gives the appearance of physical help from the variation of optical density of the sample. The DIFFERENTIAL INTERFERENCE CONTRAST MICROSCOPY converts specimen optical path gradients into amplitude differences that can be visualized as improved contrast in the resulting image. The optical components required for differential interference contrast microscopy do not obstruct the objective and condenser diaphragms as in phase or Hoffman modulation contrast, as a result enabling the instrument to be employed at full numerical aperture. The result is a dramatic improvement in resolution particularly in the direction of the optical axis, elimination of halo artifacts, and the ability to produce excellent images with relatively thick specimens.

Some advantages of interference-derived contrast Microscopy consist of making the object appear bright against a dark background but without the diffraction halo associated with phase contrast. Interference-derived contrast Microscopy also uses live and unstained biological samples, such as a smear from a tissue culture or individual water borne single-celled organisms; its resolution and clarity in conditions are unmatched among standard optical microscopy techniques. It also produces an image that can be easily manipulated using digital and video imaging techniques to further enhance contrast.

However it has also some limitations such as the requirement for a transparent sample of fairly similar refractive index to its surroundings, which is unsuitable in biology for thick samples, such as tissue slices, and highly pigmented cells. It is also unsuitable for most non biological uses because of its dependence on polarization, which many physical samples would affect. It also suffers to represent reality because DIFFERENTIAL INTERFERENCE CONTRAST MICROSCOPY utilizes optical path differences within the specimen by using product of refractive index and geometric path length to generate contrast the three-dimensional appearance, which means it is only an optical image. Read the entire article...