Unstained specimens often appear inconspicuous and depleted of details in brightfield microscopy. But actually they interact with the striking light and phase shifts occur that are not detectable with the human eye. Staining would lead to an amplitude shift and a difference in the intensity of the light passing through, but mostly this is only possible for dead material. DIC microscopy is a technique which uses gradients in the optical path length and phase shifts to make phase objects visible under the light microscope. In this way it is possible to observe living cells and organisms with adequate contrast and resolution.
Images produced by a DIC microscope are relief-like and seem to have a shadow cast. They have no halo artifacts and relatively thick specimens can also be imaged due to the possibility of optical sectioning.
In DIC microscopy, only polarized light is used to illuminate the specimen. The polarized light is dispersed into two distinct light rays with an orthogonal plane of polarization. These two light rays are extremely near to each other. As they experience different refracting or scattering in the specimen different phase shifts occur. If these light rays reunite, they will interfere with each other. The light is now elliptically polarized. This polarization can be changed into an amplitude shift via an analyzer. In this way, phase shifts of wavelength differences between up to 1/200 wavelength (or even 1/1000 wavelength using a camera) and a whole wavelength can be made visible.
In non-polarized light the light waves oscillate in all directions in respect to the axis of their propagation. In linearly polarized light beams, however, all light waves’ oscillations have the same angle or plane of polarization. Circular polarization is another possible form of polarization. The light wave’s oscillation follows a circular movement and has a stable absolute value, whereas the direction changes with a constant angular velocity. Elliptically polarized light is a mixture of linearly and circularly polarized light.
Polarized light can be produced by a device called a polarizer. An analogous component is called an analyzer if it is used to determine the plane of vibration. There are absorptive and beam-splitting polarizers.
In a DIC microscope, polarized light is produced via a polarizer in front of the condenser. This polarized light is split into two linearly polarized light rays with a perpendicular plane of polarization via a DIC prism better known as a Wollaston prism. The Wollaston prism can be found in the plane of the condenser. A standard Wollaston prism is built up of two wedges of a birefringent material that are cemented together on their base and have optic axes parallel to the outer surface and perpendicular to each other.
Polarized light passing through this prism is sheared into two closely spaced waves with different angles of deflection, the ordinary and the extraordinary wave. They have perpendicular planes of polarization and for these two waves the medium behaves as if it has a single effective refractive index. The shear of the wavefronts takes place at the interference plane that lies at the refractive index junction between the two prisms. The light waves are spatially separated by the shear angle. Their direction and the distance between them are the same for all matching light waves across the whole prism. The space between these two waves has to be smaller than the resolution limit of the microscope.
So the specimen is illuminated by closely spaced pairs of light waves that enter parallel with a lateral displacement. If these two light waves interact with specimen parts with different refractive indices or different thickness, there will be a difference in their optical path length as they emerge from the specimen. The optical path length is the product of the refractive index and the thickness between two points on an optical path. It is related to the transit time and the velocity of light. As the optical path length is related to the transit time of the light, a phase shift occurs between the two matching light waves.
After the light waves have traversed the specimen they are brought back together via another Wollaston prism. Now they interfere with each other, forming elliptically polarized light. The elliptically polarized light passes through an analyzer. Only light with a distinct polarization plane is able to pass and therefore different amplitudes are produced for the different light waves. The phase shift is transformed into an amplitude shift which leads to different light intensities in the resulting image.
For modern DIC microscopes the Wollaston prism is often modified. One example is the Nomarski prism. It consists of two birefringent wedges as well but only one wedge is identical to the one in a Wollaston prism. The other one is modified and the optical axis is obliquely positioned. This leads to an interference plane which lies outside the prism. So the prism can be located outside the objective’s aperture plane and is easier to use.
In DIC microscopy the phase shift differences of neighboring object points contribute to the image formation. Details of the specimen which have a gradient in their refracting indices or their thickness are visualized. As the light beams are polarized perpendicularly to each other different images can be produced by rotating the microscope stage.
It is important to remember not to use plastic in DIC microscopy because many polymers have a depolarizing effect on light and would destroy the contrast.
Fig. 4: Unpolarized light from a light source passes through a polarizing filter and is polarized at 45°. After passing through the Wollaston prism, the light is separated into perpendicular polarized components, one with 0° and one with 90° polarization. The condenser subsequently directs the light through the sample. The sample is illuminated by two coherent parallel light rays with different polarization. This basically produces two slightly offset brightfield images of the sample, one with 0° and one with 90° polarized light. Due to the different polarization of the light, these images do not interfere. The separated rays experience different optical path lengths, as there might be differences in the thickness or refractive index at the point they pass through the sample. This results in a phase shift of one ray compared to the other. After passing through the objective, the perpendicularly polarized light is recombined to one polarized at 135°. According to the differences in the optical path length, interference of the two rays now produces brightening or darkening, making otherwise hardly visible structures appear. Finally, directly transmitted light, which did not experience any phase shift, is removed by the polarizing filter with 135° direction (also called analyzer).
Fig. 6a-c: C. elegans recorded with differential interference contrast (DIC) and Wollaston prisms with different splitting angles