Microscopy

Nomarski (DIC) microscopy

imageNomarski or differential interference contrast (DIC) microscopy (invented by Georges Nomarski in the mid-1950s) is used to image, living or stained specimens, which contain little or no optical contrast when viewed using brightfield illumination.

In transmitted light DIC, light from the lamp is passed through a polariser located beneath the condenser, in a manner similar to polarised light microscopy. The polarised light then passes though a modified Wollaston prism (below the condenser) that splits the entering beam of light into two beams travelling in slightly different directions but vibrating perpendicular to each other, and therefore unable to recombine to cause interference. The distance between the two beams is called the "shear" distance, and is always less than the resolving ability of the objective, to prevent the appearance of double images. The split beams enter and pass through the specimen where their paths are altered by the specimen's varying thickness, slopes, and refractive indices. When the parallel beams enter the objective, they are focused above the rear focal plane where they enter a second modified Wollaston prism that recombines the two beams at a defined distance outside of the prism. This removes the shear and the original path difference between the beam pairs. However, the parallel beams are no longer the same length because of path changes caused by the specimen. In order for the parallel beams to interfere with each other, the vibrations of the beams of different path length must be brought into the same plane and axis. This is accomplished by placing a second polariser (analyzer) above the upper Wollaston beam-combining prism. The light then proceeds toward the eyepiece where it can be observed as differences in intensity and colour. DIC microscopy causes one side of an object to appear bright (or coloured) while the other side appears darker (or a different colour). This shadow effect gives a pseudo three-dimensional appearance to the specimen, but is not a true representation of the geometry of the specimen, because it is based on optical thickness. The pseudo 3D appearance of the specimen can also be profoundly affected by it's orientation i.e. rotation of the specimen by 180 degrees can change a hill into a valley or vice versa. Therefore, DIC microscopy is not suitable for accurate measurement of actual heights and depths.

These are numerous advantages in DIC microscopy as compared particularly to phase microscopy. Using DIC microscopy it is possible to make fuller use of the numerical aperture of the system because, unlike phase contrast microscopy, there is no sub-stage annulus to restrict the aperture, therefore, Köhler illumination is properly utilized. Images can be seen in striking colour (optical contrast) with a 3-dimensional shadowed-like appearance and at excellent resolution. Use of the full objective aperture also enables the microscope to focus on a thin plane section of a thick specimen without confusing images from above or below the plane (optical sectioning). There are no confusing halos, as may be encountered in phase images. DIC microscopy is superb for investigating living cells, as it is non-invasive and real-time, optical sectioning possibilities allow the movement of tiny organelles to be followed with ease. There are however, several disadvantages in DIC microscopy; the equipment for DIC is quite expensive because of the many prisms that are required. Birefringent specimens, such as those found in many kinds of crystals, may not be suitable because of their effect upon polarised light. Similarly, specimen carriers made of plastic, such as culture vessels, Petri dishes etc, may not be suitable. For very thin or scattered specimens, better images may be achieved using phase contrast methods.