Differential interference contrast microscopy

Differential interference contrast microscopy (DIC), also known as Nomarski Interference Contrast (NIC) or Nomarski microscopy, is an optical microscopy illumination technique used to enhance the contrast in unstained, transparent samples. DIC works on the principle of interferometry to gain information about the optical density of the sample, to see otherwise invisible features. A relatively complex lighting scheme produces an image with the object appearing black to white on a grey background. This image is similar to that obtained by phase contrast microscopy but without the bright diffraction halo.

DIC works by separating a polarised light source into two beams which take slightly different paths through the sample. Where the length of each optical path (i.e. the product of refractive index and geometric path length) differs, the beams interfere when they are recombined. This gives the appearance of a three-dimensional physical relief corresponding to the variation of optical density of the sample, emphasising lines and edges though not providing a topographically accurate image.

The light path


1. Unpolarised light enters the microscope and is polarised at 45°.
 * Polarised light is required for the technique to work.

2. The polarised light enters the first Nomarski-modified Wollaston prism and is separated into two rays polarised at 90° to each other, the sampling and reference rays.
 * Wollaston prisms are a type of prism made of two layers of a crystalline substance, such as quartz, which, due to the variation of refractive index depending on the polarisation of the light, splits the light according to its polarisation. The Nomarski prism causes the two rays to come to a focal point outside the body of the prism, and so allows greater flexibility when setting up the microscope, the prism can be actively focused.
 * Wollaston prisms are a type of prism made of two layers of a crystalline substance, such as quartz, which, due to the variation of refractive index depending on the polarisation of the light, splits the light according to its polarisation. The Nomarski prism causes the two rays to come to a focal point outside the body of the prism, and so allows greater flexibility when setting up the microscope, the prism can be actively focused.

''3. The two rays are focused by the condenser for passage through the sample. These two rays are focused so they will pass through two adjacent points in the sample, around 0.2 μm apart.''
 * The sample is effectively getting illuminated by two coherent light sources, one with 0° polarisation and the other with 90° polarisation. These two illuminations are, however, not quite aligned, with one lying slightly offset with respect to the other.



''4. The rays travel through the different, adjacent, areas of the sample. They will experience different optical path lengths where the areas differ in refractive index or thickness. This causes a change in phase of one ray relative to the other due to the delay experienced by the wave in the more optically dense material.''
 * The passage of many pairs of rays through pairs of adjacent points in the sample (and their absorbance, refraction and scattering by the sample) means an image of the sample will now be carried by both the 0° and 90° polarised light. These, if looked at individually, would be bright field images of the sample, slightly offset from each other. The light also carries information about the image invisible to the human eye, the phase of the light. This is vital later. The different polarisations prevent interference between these two images at this point.

5. The rays travel through the objective lens and are focused for the second Nomarski-modified Wollaston prism.

''6. The second prism recombines the two rays into one polarised at 135°. The combination of the rays leads to interference, brightening or darkening the image at that point according to the optical path difference.''
 * This prism overlays the two bright field images and aligns their polarisations so they can interfere. However the images do not quite line up because of the offset in illumination, this means that, instead of interfering with a ray of light that passed through the same point in the specimen, it will interfere with a ray of light that went through an adjacent point and have a slightly different phase. Because the difference in phase is due to the difference in optical path length this recombination of light causes "optical differentiation" of the optical path length, generating the image seen.

The image


The image has the appearance of a three dimensional object under very oblique illumination, causing strong light and dark shadows on the corresponding faces. The direction of apparent illumination defined by the orientation of the Wollaston prisms.

As explained above the image is generated from two identical bright field images being overlayed slightly offset from each other (typically around 0.2μm), and the subsequent interference due to phase difference converting changes in phase (and so optical path length) to a visible change in darkness. This interference may be either constructive or destructive, giving rise to the characteristic appearance of three dimensions.

The typical phase difference giving rise to the interference is very small, very rarely being larger than 90° (a quarter of the wavelength). This is due to the similarity of refractive index of most samples and the media they are in, for example a cell in water only has a refractive index difference of around 0.05. This small phase difference is important for the correct function of DIC, if the phase difference at the joint between two substances is too large then the phase difference would reach 180° (half a wavelength), which is complete destructive interference, and then move on toward 360° (a full wavelength), where it would reach complete constructive interference, and an anomalous bright region.

It is worth noting the image can be approximated (neglecting refraction and absorption due to the sample and the resolution limit of beam separation) as the differential of optical path length with respect to position across the sample, and so the differential of the refractive index (optical density) of the sample.

Advantages and disadvantages
DIC has strong advantages in uses involving 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 such as this are unrivaled among standard optical microscopy techniques.

The main limitation of DIC is its requirement for a transparent sample of fairly similar refractive index to its surroundings. DIC is unsuitable (in biology) for thick samples, such as tissue slices, and highly pigmented cells. DIC is also unsuitable for most non biological uses because of its dependence on polarisation, which many physical samples would affect.

Image quality, when used under suitable conditions, is outstanding in resolution and almost entirely free of artifacts. However analysis of DIC images must always take into account the orientation of the Wollaston prisms and the apparent lighting direction, as features parallel to this will not be visible. This is, however, easily overcome by simply rotating the sample and observing changes in the image.