The introduction of bias retardation in differential interference contrast (DIC) microscopy renders the specimen image in pseudo three-dimensional relief where regions of increasing optical path length (sloping phase gradients) appear much brighter (or darker), and those exhibiting decreasing path length appear in reverse. This interactive tutorial explores the effects of varying bias retardation on contrast as a function of thickness for a wide spectrum of semi-transparent specimens.
The tutorial initializes with a randomly selected image of a transparent or semi-transparent specimen in the Specimen Image window. The virtual DIC microscope is configured with a de Sénarmont compensator (consisting of a polarizer and a quarter-wavelength retardation plate) and fixed Nomarski prisms in the condenser turret and above the objective. When the specimen image first appears, the polarizer in the de Sénarmont compensator is set to zero degrees (parallel to the fast axis of the quarter-wavelength retardation plate) at maximum extinction, and no bias retardation is applied to the wavefronts traveling through the specimen.
In order to operate the tutorial, use the Bias Retardation slider to simulate addition of either positive (moving the slider to the right) or negative (moving the slider to the left) bias to the specimen image. The bias retardation range is set by the de Sénarmont compensator design, and extends from a polarizer rotation angle of -45 degrees to +45 degrees. As the slider is translated to the right and left, the effects of bias retardation applied to wavefronts passing through the specimen are displayed as increased or decreased contrast in theSpecimen Image window, and the retardation value is presented in both fractions of a wavelength and nanometers (displayed in the lower yellow box). In addition, the current polarizer angle for the de Sénarmont compensator is displayed in the upper yellow box. In order to examine another specimen, use the Choose A Specimen pull-down menu.
Presented in Figure 1 is a series of digital images recorded in DIC using a bias retardation range of one-twentieth to a quarter wavelength in several intermediate steps. The specimen is a human buccal mucosa (cheek) epithelial cell that contains regions of fluctuating thickness ranging from 1 to about 4 micrometers. Rendition of specimen detail and the shadow-cast pseudo three-dimensional effects are the most pronounced at the lower bias retardation values (Figure 1(a) and 1(b)), and both contrast and definition of fine specimen detail deteriorate as bias retardation is increased (Figure 1(c) through 1(f)). At the highest bias retardation value (one-quarter wavelength; Figure 1(f)), contrast is extremely poor and very few structural details are visible. For this particular specimen, the optimum retardation range appears to lie between one-twentieth and one-sixteenth of a wavelength.
As the optical path gradient in a specimen increases, so does image contrast. Altering the bias retardation to varying degrees can also produce significant contrast fluctuations in the specimen as observed in the eyepieces (Figure 1). In general, the optimum degree of displacement between the ordinary and extraordinary wavefronts induced by translation of the objective prism or by rotating the polarizer in a de Sénarmont compensator is on the order of less than one-tenth wavelength. However, this value is largely dependent on specimen thickness, and the useful range of bias retardation for biological specimens lies between one-thirtieth and a quarter wavelength. Contrast in specimens having very large optical gradients can often benefit from even greater bias retardation values (up to a full wavelength). The introduction of bias retardation into a differential interference contrast microscope enables phase specimens to be observed with greater ease, and dramatically facilitates imaging efforts with traditional film or digital camera systems.
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.
Kenneth R. Spring - Scientific Consultant, Lusby, Maryland, 20657.
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.