What is DIC? Why is it used in cell microscopy?
Differential interference contrast microscopy (DIC) requires plane-polarized light and additional light-shearing (Nomarski) prisms to exaggerate minute differences in specimen thickness gradients and refractive index. Lipid bilayers, for example, produce excellent contrast in DIC because of the difference in refractive index between aqueous and lipid phases of the cell. In addition, cell boundaries in relatively flat adherent mammalian and plant cells, including the plasma membrane, nucleus, vacuoles, mitochondria, and stress fibers, which usually generate significant gradients, are readily imaged with DIC. In plant tissues, the birefringent cell wall reduces contrast in DIC to a limited degree, but a properly aligned system should permit visualization of nuclear and vacuolar membranes, some mitochondria, chloroplasts, and condensed chromosomes in epidermal cells. Differential interference contrast is an important technique for imaging thick plant and animal tissues because, in addition to the increased contrast, DIC exhibits decreased depth of field at wide apertures, creating a thin optical section of the thick specimen. This effect is also advantageous for imaging adherent cells to minimize blur arising from floating debris in the culture medium.
How does DIC microscopy work?
DIC for a modern transmitted light microscope is different than that in reflected light because two birefringent prisms are used (see Figure 6), and the specimen's optical path difference is determined by the product of the refractive index difference (between the specimen and its surrounding medium) and the thickness (geometrical distance) traversed by a light beam between two points on the optical path. Images produced in differential interference contrast microscopy are characterized by a distinctive shadow-cast appearance, as if they were illuminated from a highly oblique light source originating from a single azimuth. Unfortunately, this effect, which often will render a specimen in a pseudo three-dimensional relief (Figure 2(d)), is frequently assumed by uninformed microscopists to be an indicator of actual topographical structure.
Figure 2: DIC light pathway and components.
Figure 2 illustrates the differential interference contrast beam path that is similar to that of polarized transmitted light. In DIC (as compared to polarized light), the two birefringent prisms (Figure 2(b)) are inserted into the optical train, one in the condenser and the second near the objective pupil. The condenser prism performs a vectorial decomposition of the previously linearly polarized light (Figure 2(c)) into two vibration directions that are perpendicularly polarized to each other, and laterally shifts these partial beams in such a way that a small lateral displacement of the wavefronts occurs where regions of thickness or refractive index vary. If the two partial beams now pass through exactly the same structures, no further path difference will occur in the specimen. However, if the two partial beams see slightly different conditions, each of them will experience a slightly difference pathlength that accompanies it on the remaining trip to the intermediate image plane.
As discussed, the split DIC light beams enter and pass through the specimen where their wave paths are altered in accordance with the specimen's varying thickness, slopes, and refractive indices. These variations cause alterations in the wave path of both beams passing through areas of any specimen details lying close together. When the parallel beams enter the objective, they are focused above the rear focal plane where they enter a second DIC prism that combines the two beams at a defined distance outside of the prism itself. This removes the shear (distance between the waves) and the original path difference between the beam pairs. As a result of having traversed the specimen, the paths of the parallel beams are not of the same length (optical path difference) for differing areas of the specimen.
In order for the beams to interfere, 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 polarizer (analyzer) above the upper DIC beam-combining prism. The light then proceeds toward the eyepiece where it can be observed as differences in intensity and color. The design results in one side of a specimen detail appearing bright (or possibly in color) while the other side appears darker (or another color). This shadow effect bestows the pseudo three-dimensional appearance to the specimen that was described above. There are numerous advantages in DIC microscopy as compared to phase contrast microscopy. With DIC, it is possible to make fuller use of the numerical aperture of the system and to provide optical staining (color). DIC also allows the microscope to achieve excellent resolution. Use of full objective aperture enables the microscopist to focus on a thin plane section of a thick specimen without confusing images from above or below the plane. Annoying halos, often encountered in phase contrast, are absent in DIC images, and suitable achromat and fluorite objectives can be used for this work. It is important to keep in mind, however, since DIC is based on polarized light principles, highly birefringent specimens or those embedded in birefringent materials should not be observed under DIC.
Setting up a DIC microscope:
Follow the steps described HERE.
References:
http://zeiss-campus.magnet.fsu.edu/articles/basics/contrast.html
Sanderson, 2007: Setting up the Differential Interference Contrast (DIC) microscope