DIFFERENTIAL INTERFERENCE CONTRAST (DIC) MICROSCOPY

 DIFFERENTIAL INTERFERENCE CONTRAST (DIC) MICROSCOPY

·       Many biological specimens cannot be effectively visualized using ordinary Bright field  microscopy because their images produce very little contrast, rendering them essentially invisible.

·       Differential Interference Contrast (DIC) microscopy is an excellent microscopy technique that introduces contrast to images of specimens which have little or no contrast when viewed using Brightfield microscopy.

·       Differential Interference contrast (DIC) microscopy was invented by Francis Smith in 1947 and further developed by the French Physicist Georges Nomarski in the 1952 as an improvement over Phase contrast microscopy.  DIC is sometimes referred to as Nomarski microscopy.

·       Differential Interference contrast (DIC) microscopy is best for visualizing unstained samples.

·       Images produced using Differential Interference Contrast (DIC) optics have a distinct relief-like, shadow-cast appearance giving an illusion of Three dimensionality (3D).

Advantages of DIC over other Contrast techniques

·       An advantage of using DIC over other contrast techniques, such as Phase contrast or Oblique contrast is that in DIC the full aperture of the Microscope is used.

·       For example, in Phase contrast, the Annulus of the Condenser restricts the Aperture, reducing the Resolution of the image. Unlike Phase contrast, DIC images are not disturbed by halo artefacts.

·       Some of the advantages of Differential Interference contrast (DIC) microscopy over other Contrast techniques are

ü  Produces high resolution images.

ü  Shows good contrast.

ü  Can be used with thick Specimens.

ü  Lacks the distracting halo of Phase contrast.

ü  Can be further processed (Video enhanced).

Parts of Differential Interference contrast (DIC) microscopy

·       Polarizer - Inserted in microscope train between the incandescent illumination source and the condenser, this component is designed to produce linearly polarized or plane-polarized light necessary for the interference detection. To provide more image contrast adjustment, some differential interference contrast kits use a rotating polarizer and a quarter-wavelength retardation plate. When the contrast is perfectly adjusted the image of the specimen will have a three dimensional effect.

·       Nomarski Prism or Condenser prism or Wollaston prism – It is a beam-splitting prism divides the polarized light beam emanating from the polarizer into two beams. If this prism is separate from the condenser, the light beams are transmitted to the condenser; however it is often incorporated into the design of a specialized condenser. These light beams are known as ordinary and extraordinary or specimen and reference beams.

·       Specimen slides and vessels - One important restriction of DIC is that plastic vessels cannot be used due to the strain they exhibit under crossed polars. For upright microscopes this is not an issue, but users of inverted microscopes may consider using plastic vessels which have a glass insert of coverslip thickness – these are available commercially.

·       Objective - Theoretically any Objectives can be used, but in practice higher grade Objectives (Fluorite and apochromatic types) are generally specified to benefit from the high-resolution potential. In many cases phase-contrast fluorite objectives are chosen, permitting Brightfield, DIC, Phase contrast and Fluorescence observation with a single set of objectives.

·       Objective prism or Objective specific prism - Either adjustable or fixed mounted, this upper prism recombines the separated beams into elliptically polarized light. Like the lower prism, this prism is formed by affixing two optical quartz wedges together. The wedges are cut differently so that one of them has its optical axis parallel to the prism’s surface and the other one’s optical axis is at an angle to the prism’s flat surface.

·       Analyzer - Located behind the objective prism and is oriented perpendicular to the transmission path of the lower polarizer. This is where the interference occurs that generates the differential interference contrast.

·       The light that passed through the specimen will have a different refractive index occasioned by the differences in the thickness of the different structures and areas of the specimen.

·       Interestingly, if no specimen is in place and both light beams enter the upper prism without any refractory differences, the effect of the lower prism is exactly reversed by the upper prism and the image field appears black, an effect known as extinction.

Working Principle of Differential Interference contrast (DIC) microscopy

Differential Interference contrast (DIC) microscopy

·       Light passes through a standard polarizer before entering the condenser, producing plane-polarized light.

·       This light enters a Wollaston prism (DIC prism) situated in the front focal plane of the Condenser. The prism interacts with the polarized light to produce two separate wavefronts polarized perpendicularly to each other. These are termed the Ordinary (O) and Extraordinary (E) rays. Furthermore, these two wavefronts are separated by a very small difference (less than the resolution of the system). This separation is termed Shear and is an important characteristic of the system.

·       The two wavefronts pass through the specimen, and are retarded to varying extents in doing so.

·       The light now enters a second Wollaston prism (DIC prism) set-up which recombines the wave fronts. If there has been a phase shift between the two rays as they pass through areas of different refractive index then elliptically polarized light is the result.

·       Finally, the light enters a second polarizing filter, termed an analyzer. The initial polarizer and this analyzer form crossed polars. The analyzer will permit the passage of some of the elliptically polarized light to form the final image. All the remaining light will be blocked by the Analyzer.

Applications of DIC

·       Visualizing Unstained Specimens - Like phase contrast, DIC is a very useful tool for visualising unstained specimens. This is clearly an advantage when observing living specimens, such as small organisms, tissues or cells. In addition to simply observing such specimens DIC can be used effectively in several other specialised applications listed below.

·       Locating the specimen or even the focal plane using fluorescence illumination can be a challenge. DIC, like phase contrast, can be used at low illumination levels for this task, but more importantly for indication where in the specimen a labelled component resides.

·       Infrared DIC - One interesting application of DIC is the imaging of cells inside tissues, such as brain slices used in electrophysiology. Here, infrared (IR) light is used as it penetrates deeper into the tissue slice than visible light. Appropriate optics with high IR transmission must be utilised, and the image is captured using an infrared camera. To the untrained eye these IR-DIC images look blurred but they are highly valued by neurophysiologists. A further advantage of this technique is that the microscope can be equipped with a sensitive camera to capture the fluorescence image (often of very short duration) while the IR camera gives structural information.

·       DIC is used in Metallurgy, Materials and Semiconductors, producing good images of surface features such as scratches.

Advantages of DIC

·       An advantage of DIC is that the specimen will appear bright in contrast to the dark background.

·       This method can take advantage of being able to use a full width condenser aperture setting. Where originally a slit condenser had to be used to produce a thin vertical beam of light, this limited the amount of illumination that could be brought to focus on the specimen.

·       The lower prism allows the user to employ the full condenser aperture by compensating for the phase differences of all the emitted light and results in a brighter image.

·       This system is relatively easy to incorporate with an existing brightfield microscope. Two of the short comings of the phase contrast method are the fact that the specimen must be very thin and a halo is produced in the viewing field.

·       No halo effect occurs with differential interference contrast and it can be used to produce very clear images of thick specimens.

·       It can also be used in conjunction with digital imaging systems to add further definition to the image.

·       Differential interference contrast imaging can be used in conjunction with fluorescence microscopy to provide a better fluorescence image and to pinpoint specific areas on a specimen before switching to the fluorescence mode to further examine the object.

·       A major advantage of the differential interference contrast technique is in examining living specimens when normal biological processes might be impeded by normal staining procedures.

Disadvantages of DIC

·       A drawback to this type of imaging is that the three-dimensional image of a specimen may not be accurate.

·       The enhanced areas of light and shadow might add distortion to the appearance of the image.