In reflected light differential interference contrast (DIC) microscopy, the spatial relationship and phase difference between ordinary and extraordinary wavefronts passing through the optical system is governed either by the position of the objective prism (Nomarski DIC) or the orientational relationship between the polarizer and a thin quartz retardation plate in a de Sénarmont design. This interactive tutorial explores the similarities and differences between the wavefront relationships in the two microscope configurations.
The tutorial initializes with an animated on-axis wavefront passing through a traditional reflected light Wollaston DIC microscope optical train. Although the tutorial presents a Wollaston prism configuration for ease of illustration, the same effects are observed with a Nomarski prism. To begin the optical pathway, a beam of linearly polarized light emerges from the polarizer (upper right-hand corner of the tutorial) and is reflected by a half-mirror through the Wollaston prism (labeled Objective Prism), where it is sheared into two orthogonal components. In the tutorial, the separation distance between the sheared wavefronts is greatly exaggerated to enhance visualization. After passing through the objective, reflecting from the surface of the specimen, and then traveling back to the prism along the same route, the orthogonal wavefronts are recombined before proceeding upward through the half-mirror.
The emerging waveform (leaving the Wollaston prism) is indicated by a line, ellipse, or circle in the space between the half-mirror and the analyzer. When the waveform traveling up the optical system through the half-mirror is linear, no light is passed through the analyzer, which is signified by the Wavefront Blocked message. However, elliptical and circular wavefronts (which result from an optical path difference due to either the introduction of bias retardation or the specimen) are able to pass a component through the analyzer to form an image (indicated by the Wavefront Passed text above the analyzer). Bias retardation can be introduced into the system by translating the Objective Prism Position slider, which shifts the Wollaston prism back and forth across the virtual microscope optical axis and affects the character of the emerging waveform. Similarly, introducing a small gradient on the specimen surface with the Specimen Gradient slider also affects the optical path difference relationship between orthogonal wavefronts reflected from the specimen and produces elliptically or circularly polarized light, which can pass a linear component through the analyzer.
The corresponding optical pathway for off-axis rays can be examined by activating the Off-Axis radio button in the Light Path collection near the lower left-hand corner of the tutorial window (On-Axis is the default setting). The speed of wavefronts passing through the virtual microscope optical train can be controlled with the Applet Speed slider. The microscope DIC configuration can be toggled between Wollaston and de Sénarmont with the Imaging Mode radio button set in the lower right-hand corner of the tutorial window. In all tutorial operating modes, the incident ordinary wavefront is represented by short blue lines (parallel to the browser window) and the extraordinary wavefront is represented by green dots (perpendicular to the browser window). Likewise, the reflected ordinary wavefront is represented by short red lines, while the extraordinary wavefront is depicted by black dots.
In de Sénarmont DIC imaging mode, a quarter-wavelength retardation plate is inserted into the reflected light optical pathway between the polarizer and the half-mirror. When the polarizer orientation is parallel to the fast axis of the retardation plate (default position), linearly polarized light emerges from the de Sénarmont compensator and is reflected from the half-mirror before entering the Wollaston prism. As the polarizer is rotated either in a positive or negative direction (using the Polarizer Rotation Angle slider), the wavefronts emerging from the de Sénarmont compensator are split into orthogonal components and have an optical path difference that is a function of the polarizer rotation angle. In this manner, bias retardation is introduced into the reflected light de Sénarmont DIC optical system. Other characteristics of the optical pathway, as available with Nikon's LV100N POL microscope are similar to the traditional reflected light DIC configuration.
Chris Brandmaier - Industrial Microscope Division, Nikon Instruments, Inc., 1300 Walt Whitman Road, Melville, New York 11747.
Kenneth R. Spring - Scientific Consultant, Lusby, Maryland, 20657.
Robert T. Sutter and Michael W. Davidson - National High Magnetic Field Laboratory, 1800 East Paul Dirac Dr., The Florida State University, Tallahassee, Florida, 32310.