Unlike phase contrast, which is based on total refractive index, DIC is based on the local change in refractive index. This contrast enhancing method also allows visualisation of unstained samples, eg. unstained tissue or cells, but is suitable for thicker samples than phase contrast. It is based on light refraction differences of different parts of the transparent specimen. This effect is achieved by making each ray of light interfere with another passing through the specimen a very small distance away from it.

If the refractive index of the specimen is changing, there will be a path difference between the two rays, if it is uniform, there won’t be.

The contrast we see in the final image will depend on the local rate of change of refractive index in the specimen - hence the name differential interference contrast.

DIC is often used as an overlay channel with fluorescent labels and tends to give a somewhat 3D effect. It is also widely used in microinjection techniques where it is necessary to see clearly defined, intracellular structures such as nuclei.

DIC will only work through glass, so if you want to use this technique with living, cultured cells, you must set up your cultures in dishes with glass bottoms and lids.

DIC image of Spirogyra by Dr Louise Cole

DIC of a breast cancer cell in a collagen matrix by Sandra Fok

Hela cells in culture stained with Hoechst 33342 to label nuclei and imaged with both fluorescence and DIC. There are a range of Hoechst stains that be used with living cells. By Ying Ying Su.

Heart muscle visualised using confocal microscopy superimposed with differential interference contrast microscopy by Dr Paul Monaghan, CSIRO.

The microscope is set up as shown in the diagram

• A polariser sits below the condenser to generate a beam of polarised light.
• A Wollaston prism sits at the position of the condenser aperture. This is made of two quartz wedges, with their crystal axes at right-angles, cemented together. Quartz is birefringent so it will resolve the incoming polarised light into two rays emerging at slightly different angles.
• The λ/4 plate puts a quarter wave path difference between them. This can be either above or below the objective-specific Wollaston prism.
• The condenser lens will focus these two rays to slightly different points on the specimen. These points must be closer together than the resolution of the microscope, so that you don’t see a double image.
• As the rays emerge from the sample, they are recombined in a second Wollaston prism past the objective although the two rays will have different lengths from passing through different parts of the sample. This recombined beam is then depolarised by passage through a second polariser, also called an analyser.
• Positive or negative bias can then also be introduced to cause features to appear as either raised or depressed. This can be very valuable for optimising the appearance of particular features of interest.

A ‘relief’ effect – striking but entirely artificial