Although in traditional designs, differential interference contrast (DIC) microscopes introduce bias retardation into the matched condenser and objective Nomarski (or Wollaston) prisms by translating one of the prisms across the optical axis, the same effect can also be achieved through the use of a simple de Sénarmont compensator with fixed Nomarski prisms. This interactive tutorial examines the relationship between wavefronts emerging from a de Sénarmont compensator and how they can be controlled to produce positive and negative bias retardation (contrast) effects in a DIC microscope.
The tutorial initializes with a cutaway model of a de Sénarmont compensator designed to fit a Nikon upright microscope appearing in the window. A beam of non-polarized light is incident on the normal plane of the polarizing element in the compensator, and passes through the linear polarizer before impacting on the quarter-wavelength plate at a 45-degree angle to the fast and slow axes. The polarized light, thus oriented, emerges from the compensator in the form of two orthogonal wavefronts that combine to produce right-handed circularly polarized light. In the tutorial, polarized light waves oriented parallel to the fast axis of the compensator (indicated by a red wedge on the plate) are represented as blue sine waves, while those polarized waves oriented parallel to the compensator slow axis (black wedge on the plate) are depicted as red sine waves.
The pathway of circularly polarized light exiting the compensator is calculated by the vector summation of the orthogonal wave components, and is illustrated in the tutorial as a black arrow that traces a circular arc around the periphery of the wavefronts. In all cases, the sine waves emerging from the de Sénarmont compensator are exaggerated in size to more clearly demonstrate their relationship to one another and to the compensator axes. The entire compensator assembly can be rotated within the tutorial window by placing the mouse cursor on any component, and then dragging the model to a new position.
In order to operate the tutorial, use the Polarizer Orientation slider to change the orientation of the polarizer with respect to the quarter-wavelength retardation plate in the compensator. As the slider is translated from the default position (+45 degrees), the phase relationship between orthoganol wavefronts is altered, and emerging polarized light changes from being circularly polarized to having varying degrees of elliptical polarization. These effects are most clearly observed when the top of the compensator model is dragged to the front of the window and the wavefronts are viewed end-on.
When the polarizer transmission axis becomes perfectly aligned (parallel) with the fast axis of the retardation plate (in this case, the Polarizer Orientation slider is set to zero degrees), only linear light emerges from the de Sénarmont compensator and no bias retardation is introduced into the optical system. Moving the slider further to the left (negative values) produces left-handed elliptically polarized light at polarizer orientations between 1 and 44 degrees, and circularly polarized light again at -45 degrees. The total amount of bias retardation introduced into the system is constantly updated as the Polarizer Orientation slider is translated, and the current value is presented in the yellow box positioned in the upper left-hand corner of the tutorial window.
Translation of the sine wave(s) can be halted by removing the check mark in the Translate Sine Wave checkbox. The Applet Speed slider controls the speed of the sine wave(s) passing through (and emerging from) the compensator, and should be used to slow the progression of wavefronts for observation. Clicking the Pause button will freeze the tutorial in the current configuration, but still enable rotation of the model through all three dimensions. Clicking the Reset button will return the tutorial to its initialization state.
In a majority of the traditional DIC microscope configurations, bias retardation is introduced by translating the objective Nomarski prism back and forth along the optical axis using a fine adjustment (micrometer) knob located at the end of the mounting frame (which is usually positioned in the microscope nosepiece housing or an intermediate tube). An alternate and more quantitative technique, termed the de Sénarmont DIC compensation method and now utilized by Nikon and other manufacturers, employs a quarter-wavelength retardation plate in fixed orientation between the polarizer and condenser prism. At maximum extinction, the fast axis of the retardation plate is aligned with the transmission axis of the polarizer, and both optical units can be (and often are) contained within the same housing on the base of an upright microscope or above the condenser for an inverted microscope. An alternative location for the de Sénarmont compensator, in microscopes equipped with the appropriate intermediate tube, is between the objective prism and the (rotatable) analyzer.
In order to introduce bias retardation using the de Sénarmont compensator, the polarizer transmission axis is rotated (up to plus or minus 45 degrees) with respect to the fast axis of the retardation plate, which remains fixed at a 90-degree angle relative to the analyzer transmission axis. When the compensator fast axis coincides (is parallel) with the transmission axis of the polarizer, only linearly polarized light passes through the de Sénarmont compensator to the condenser Nomarski prism (as illustrated in Figure 1(b)). However, when the polarizer transmission axis is rotated, wavefronts emerging from the quarter-wavelength retardation plate in the de Sénarmont compensator become elliptically polarized (depending upon the orientation) as presented in Figures 1(a) and 1(c). Rotating the polarizer in one direction (positive) will produce right-handed elliptically polarized light, while rotating the polarizer in the other (negative) direction will alter the vector trajectory to generate a left-handed elliptical sweep.
When the orientation of the polarizer transmission axis reaches either plus or minus 45 degrees (equivalent to one-quarter wavelength of retardation), light passing through the de Sénarmont compensator becomes circularly polarized (again, in either a left-handed or right-handed sense). Because elliptically or circularly polarized light represents a phase difference between the ordinary and extraordinary wavefronts emerging from the de Sénarmont compensator, bias retardation is introduced to the system when the wavefronts enter the Nomarski prism (and become sheared) located in the microscope condenser. Positive bias is obtained when the polarizer is rotated in one direction, while negative bias is introduced by rotating the polarizer in the opposite direction.
Regardless of whether bias is introduced into a differential interference contrast system by translating the objective Nomarski prism or by rotating the polarizer on a de Sénarmont compensator, the net result is the same. In a properly configured microscope that is aligned for Köhler illumination, an image of the light source and condenser prism is transferred by the optical system (condenser and objective) onto the inverted second Nomarski prism located at the objective rear focal plane. The linear phase shift across the face of the condenser prism is precisely compensated by an opposite phase shift in the objective prism. Translation of the objective prism along the shear axis does not alter the phase shift distribution, but instead, adds or subtracts a constant phase difference across the entire microscope aperture. In the same manner, rotating the polarizer in a de Sénarmont compensator also introduces a variable and controlled phase difference. The matched prism system enables image formation to occur with the same bias retardation for every wavefront pair projected from the condenser aperture, irrespective of the route through which it traverses the specimen to reach the objective.
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 and Michael W. Davidson - National High Magnetic Field Laboratory, 1800 East Paul Dirac Dr., The Florida State University, Tallahassee, Florida, 32310.