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Differential Interference Contrast

Section Overview:

An excellent mechanism for rendering contrast in transparent specimens, differential interference contrast (DIC) microscopy is a beam-shearing interference system in which the reference beam is sheared by a minuscule amount, generally somewhat less than the diameter of an Airy disk. The technique produces a monochromatic shadow-cast image that effectively displays the gradient of optical paths for both high and low spatial frequencies present in the specimen. Those regions of the specimen where the optical paths increase along a reference direction appear brighter (or darker), while regions where the path differences decrease appear in reverse contrast. As the gradient of optical path difference grows steeper, image contrast is dramatically increased.

Review Articles

  • Brief Overview of DIC Microscopy

    In the mid-1950s, a French optics theoretician named Georges Nomarski modified the Wollaston prism used for detecting optical gradients in specimens and converting them into intensity differences.

  • Fundamental Concepts in DIC Microscopy

    Through a mechanism different from phase contrast, differential interference contrast converts specimen optical path gradients into amplitude differences that can be visualized as improved contrast in the resulting image.

  • DIC Microscope Configuration and Alignment

    DIC components can be installed on virtually any brightfield transmitted, reflected, or inverted microscope, provided the instrument is able to accept polarizing filters and the specially designed condenser and objective prisms.

  • Comparison of Phase Contrast and DIC Microscopy

    The most fundamental distinction between differential interference contrast and phase contrast microscopy is the optical basis upon which images are formed.

  • Fluorescence and DIC Combination Microscopy

    Fluorescence microscopy can be combined with contrast enhancing techniques, such as differential interference contrast (DIC) and phase contrast illumination, to minimize the effects of photobleaching.

Interactive Java Tutorials

  • Optical Path Gradients and Amplitude Profiles

    Explore the intricate relationship between optical path gradients and amplitude (intensity) profiles for a variety of semi-transparent specimens such as a mouse embryo in this interactive java tutorial.

  • Origin and Variation of Image Contrast

    Explore how variations in the level of bias retardation introduced by a Nomarski or Wollaston prism affect the optical path difference and resulting specimen intensity in a DIC microscope in this tutorial.

  • The Interference Background Image

    Discover how changes to the optical path difference between orthogonal wavefronts can produce a wide spectrum of interference colors that are useful in determining path lengths and for the optical staining.

  • Specimen Orientation Effects on DIC Images

    Students can explore through various specimen how image amplitude (intensity) fluctuates as the specimen orientation is varied with respect to the microscope shear axis in this interactive java tutorial.

  • Wavefront Shear in Wollaston and Nomarski Prisms

    Examine differences between the location of the interference plane in Wollaston and Nomarski prisms, and how positioning can be varied with changes.

  • DIC Wavefront Relationships and Image Formation

    Explore wavefront spatial relationships in the DIC microscope optical train, and how these relationships affect image formation in this interactive java tutorial.

  • Optical Staining with DIC Microscopy

    Choose from a list of specimen and discover how varying the amount of bias retardation can affect the appearance and level of optical staining achieved in the specimen image in this interactive java tutorial.

  • Optical Sectioning in DIC Microscopy

    Discover optical sectioning in DIC microscopy utilizing a wide spectrum of specimens (ranging from butterfly wing scales to living retina tissue) having varied thickness in this interactive java tutorial.

  • de Sénarmont Compensators

    Learn about de Sénarmont Compensators by exploring the relationship between wavefronts emanating from the compensator as the polarizer is rotated through its useful range in this interactive tutorial.

  • Wavefront Fields in DIC Microscopy

    Explore the wavefront relationships involving polarized and orthogonal wavefront components in both de Sénarmont and traditional Nomarski optical configurations in this interactive tutorial.

  • Optical Sectioning with Phase Contrast and DIC

    Explore and compare optical sectioning of thick specimens with DIC and phase contrast, and reveals the benefits of unrestricted aperture effects on obtaining well-defined sections.

  • DIC Microscope Components and Imaging Mechanisms

    See the optical orientation and sequential positioning of the DIC microscope optical components as well as the images obtained when the objective Nomarski prism is translated.

Digital Image Galleries

Differential Interference Contrast Digital Image Gallery

Thin unstained, transparent specimens are excellent candidates for imaging with classical differential interference (DIC) microscopy techniques over a relatively narrow range (plus or minus one-quarter wavelength) of bias retardation. The digital images presented in this gallery represent a wide spectrum of specimens, which vary from unstained cells, tissues, and whole organisms to both lightly and heavily stained thin and thick sections. In addition, several specimens exhibiting birefringent character are included to demonstrate the kaleidoscopic display of color that arises when anisotropic substances are imaged with this technique.

Selected Literature References and Glossary of Terms

  • Glossary of Common Terms in DIC Microscopy

    The complex nomenclature of DIC microscopy is often confusing to beginning students and seasoned microscopists alike. The resources provided are reference tools for those exploring the spectrum of topics in DIC.

  • Selected DIC Microscopy Literature References

    A number of review articles on DIC microscopy have been published by leading researchers in the field, and were utilized as references to prepare discussions.

Contributing Authors

Douglas B. Murphy - Department of Cell Biology and Anatomy and Microscope Facility, Johns Hopkins University School of Medicine, 725 N. Wolfe Street, 107 WBSB, Baltimore, Maryland 21205.

Edward D. Salmon - Department of Cell Biology, The University of North Carolina, Chapel Hill, North Carolina 27599.

Kenneth R. Spring - Scientific Consultant, Lusby, Maryland, 20657.

Mortimer Abramowitz - Olympus America, Inc., Two Corporate Center Drive., Melville, New York, 11747.

Maksymilian Pluta - Physical Optics Department, Institute of Applied Optics, 18 Kamionkowska Street, Warsaw, Poland, 03-805.

Matthew Parry-Hill, Robert T. Sutter and Michael W. Davidson - National High Magnetic Field Laboratory, 1800 East Paul Dirac Dr., The Florida State University, Tallahassee, Florida, 32310.

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