Senarmon compensators in DIC-microscopy

In conventional microscopes operating by the differential interference contrast (DIC) the bias retardation method takes place in the optical system by means of translation of one of the matched (with a condenser or an objective) prisms –  Nomarski ormodified Wollaston prism (hereinafter - WP), which are placed perpendicular to the visual axis of the microscope to create a difference in optical path length. The same effect can be obtained by using a fixed Nomarski prism and a Senarmon compensator which consists of a quarter-wave plate combined with a polarizer or an analyzer.

The polarizer and the quarter-wave plate are installed in a common housing, which is attached to the main light port of the microscope by a setscrew. After positioning, the retardation plate remains in a preliminarily set position, whereas the polarizer can be rotated through 90 degrees (+ 45 degrees) around the optical axis of the microscope. Depending on the orientation of the polarizer in relation to the retardation plate, the Senarmon compensator may illuminate the microscope optical system with linear, elliptical or circularly polarized light.

Polarized wavefront emerging from the Senarmon compensator first reaches the fixed Nomarski prism installed in the condenser unit, then it is redirected and sheared into orthogonal components (an ordinary and an extraordinary wavefront) with oscillations at a 45-degree angle to the polarized light emerging from the Senarmon compensator. The lens system of the condenser focuses the sheared wavefronts into parallel rays and projects them onto the sample. The light coming from the sample is collected by the objective and focused onto the interference plane of the second Nomarski prism, which is located in the nosepiece of the microscope. The abovementioned second Nomarski prism is matched to the condenser prism and it combines the sheared rays into coaxial orthogonal components. Although linearly polarized light emerging from the second Nomarski prism is blocked by the analyzer, elliptical or circularly polarized light can pass the components of the optical system and form an image of the sample.

The differential interference microscopy method was devised and introduced by Francis Smith in 1955. Smith designed a modified polarizing microscope and installed WP in the front focal plane of the condenser and in the rear focal plane of the objective. Due to the structural limitations of the WP, they were subsequently replaced with a more advanced system introduced by a French scientist Georges Nomarski. In the Nomarski system the prisms are physically located at some distance from the conjugate aperture planes of the condenser and the objective. Such modification allows to use the optical components of a standard microscope in modern DIC systems, which are currently widely used.

Similar to the phase contrast method, DIC-microscopy is very useful for visualizing living cells and other transparent unstained samples, which are difficult to monitor in the traditional brightfield illumination, even with the full aperture and the maximum resolution of the microscope optical system. Among other things, DIC-microscopy is not affected by such artefacts as halo effect masked apertures inherent with phase contrast optics, and enables to obtain excellent-quality images of comparatively thick samples. Moreover, the contrast of DIC-images can be easily enhanced with the help of digital video techniques. 

The effect of the differential interference contrast is in converting the optical path length difference in the difference in the sample amplitudes, which can be observed as an image of enhanced contrast in the microscope eyepieces (or recorded on film or digitally). The major determinants of the optical path length in a sample is the difference between the refractive indices of the sample and the environment, as well as the geometric distance, which are traversed by the sheared wavefronts between two points of the optical path. Images obtained with the help of DIC-system have a “shaded” appearance and seem pseudo-three-dimensional, as if they were obtained under an illumination from a single azimuth, very oblique light source. In general, the DIC-method is helpful in determining the phase gradients orientation and in using the full aperture of the objective for obtaining thin optical sections of samples, which are not obscured by elements placed beyond the intermediate focal plane.