Living cells and other transparent, unstained specimens are often difficult to observe under traditional brightfield illumination. Phase Contrast, first developed by Frits Zernike in the 1930s, is often used to images of challenging specimens, but the technique suffers from some features and cannot take advantage of the full condenser and objective apertures.
Differential Interference Contrast Microscope
-Also known as Nomarski Interference Contrast (NIC) or Nomarski microscopy.
-Is an optical microscopy technique used to enhance the contrast in unstained, transparent samples.
-DIC works on the principle of interferometry to gain information about the optical path length of the sample, to see otherwise invisible features.
-A relatively complex optical system produces an image with the object appearing black to white on a grey background.
-The technique was developed by Polish physicist Georges Nomarski in 1952
How does it work?
•A plane-polarized wavefront created by the action of the condenser and polarizer passes through the specimen.
•Local optical path differences within the specimen., caused by differences in index of refraction, create a phase distorted wavefront. Phase distortion is represented by the displaced portion of the wavefront.
•Wavefront is collected by the objective lens and is "sheared" into two offset wavefronts by the action of the upper Wollaston prism . Due to the optical path difference between the ray-pairs as they traverse different portions of the upper prism. No interaction yet occurs between the wavefronts as they are vibrating at right angles to each other.
•The wavefronts then pass through the Analyzer where they are recombined into one vibrational plane. It is at this point where constructive or destructive wave interference occurs. This causes optical path differences within the sample to be manifested as light or dark areas in the image.
•Where no interaction occurs between ray-pairs, interference of equally phased rays produces a uniform gray background.
Advantages
•An advantage of DIC is that the specimen will appear bright in contrast to the dark background.
•This system is relatively easy to incorporate with an existing brightfield microscope.
•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.
•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.
Importance
This technique gives a greater depth of focus allowing thicker specimens to be observed under higher magnifications. Producing a monochromatic image, areas of the object appear brighter as the optical path of the transmitted light increases and darker in areas of decreased length of optical path.
