Question

1. What four components are added to a transmitted light microscope to make it a DIC (differential interference contrast (DIC) microscopy ) microscope?

2. How do you adjust the bias on a DIC (differential interference contrast (DIC) microscopy ) microscope?

3. Although the images produced by a DIC (differential interference contrast (DIC) microscopy )microscope appear to be three-dimensional, they do not actually give real 3D information. Explain why.

4. Why is it important that all glass and plastic-ware used on or with the DIC (differential interference contrast (DIC) microscopy )microscope is strain-free?

5. What are the advantages of DIC (differential interference contrast (DIC) microscopy ) microscopy over phase contrast microscopy?

6. What are the advantages of phase contrast microscopy over DIC (differential interference contrast (DIC) microscopy ) microscopy?

Answer 1

Ans 1: Four basic components areadded to a transmitted light microscope to make it a DIC microscope:

a) Linear Polarizer - Inserted into the optical pathway between the microscope light port (or anywhere after the illumination source collector lens) and the condenser lens assembly, this component is designed to produce the necessary plane-polarized light for interference imaging. The vibration plane transmission axis for the electric vector component is oriented in an East-West direction (right to left when standing in front of the microscope), typical of a standard polarized light microscope.

b) Condenser Wollaston or Nomarski Prism - In order to separate the polarized light emanating from the polarizer into two components, a specialized beamsplitting prism ( condenser prism) is placed in or near the conjugate focal plane of the condenser iris diaphragm aperture. Incident wavefronts of plane-polarized light are split into mutually orthogonal polarized components by the Wollaston or Nomarski prism.

c) Objective Nomarski Prism - Positioned behind the objectiv, either in an adjustable sliding frame or a fixed mount, a second beamsplitting prism is employed to recombine the sheared wavefronts in the conjugate plane of the objective rear aperture. This component, which is an element critical to interference and image formation, is also termed the objective prism.

d) Analyzer - A second linear polarizer is installed behind the objective prism, usually in an intermediate tube between the microscope nosepiece and observation (eyepiece) tubes. Termed an analyzer, this polarizing element is positioned in the optical pathway before the tube lens (for infinity-corrected microscopes) and image plane. The analyzer is oriented with the transmission axis of the electric field vector perpendicular (North-South) to that of the substage polarizer. Components of circular and elliptically polarized light arriving from the objective prism pass through the analyzer and subsequently undergo interference to generate the DIC image at the microscope intermediate image plane.

Ans 2: In traditional differential interference contrast (DIC) microscope system designs, bias retardation is introduced into the optical train by translating one of the matched (condenser and objective) Nomarski or modified Wollaston prisms across the optical axis of the microscope to produce a constant optical path difference. The same effect can also be achieved through the application of a fixed Nomarski prism system and a simple de Sénarmont compensator consisting of a quarter-wavelength retardation plate in conjunction with either the polarizer or analyzer.

Ans 3: In DIC microscopy, regions of increasing optical path difference (sloping phase gradients) appear much brighter (or darker), and those exhibiting decreasing path length appear in reverse. Specimen features appear similar to elevated plateaus or sunken depressions depending on the phase gradient orientation, which is a distinguishing feature of differential interference contrast. However, the three-dimensional appearance only corresponds to phase gradients and not because of the actual specimen geometry.

Ans 4: It is important to use all strain-free glass and plastic-ware with the DIC microscope because it will enable their use in polarized light and differential interference contrast observation which dramatically improves the image quality and resolution at higher magnifications.

Ans 5: A primary advantage of differential interference contrast over phase contrast is

a) The ability to utilize the instrument at full numerical aperture without the masking effects of phase plates or condenser annuli, which severely restrict the size of condenser and objective apertures.

b) Improved axial resolution, particularly with respect to the ability of the DIC microscope to produce excellent high-resolution images at large aperture sizes.

Ans 6: a) The capacity to observe living cells and, as such, the ability to examine cells in a natural state.

b) Observing a living organism in its natural state and/or environment can provide far more information than specimens that need to be killed, fixed or stain to view under a microscope.

c) High-contrast, high-resolution images.

d) Ideal for studying and interpreting thin specimens.

e) Ability to combine with other means of observation, such as fluorescence.