Differential Interference Contrast (DIC) Microscopy

Ockenga, Wymke; DeRose, James; Greb, Christoph

April 27, 2023

Differential Interference Contrast (DIC) Microscopy

Making visible unstained, transparent structures of biological specimens which are difficult to see with brightfield illumination

Neurons imaged with DIC contrast. Neurons_imaged_in_DIC.jpg

How differential interference contrast (DIC) is a good alternative to brightfield illumination when using microscopy to image transparent, unstained biological specimens is explained in this article. Brightfield images of specimens can be often devoid of structure and detail, as there is little change in intensity from local absorption of light. Staining does induce a difference in the light intensity after passing through the specimen, but often stains can be toxic. DIC microscopy exploits gradients in the optical path length and phase shifts to make transparent structures visible.

Relief-like images with polarized light

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. Specimens or their structures which cause a light-wave-amplitude change are called amplitude objects [1].

Differential interference contrast (DIC) microscopy uses gradients in the optical path length and phase shifts of light to make phase objects visible when viewed with a microscope [2]. Specimens or their structures which cause a phase shift as light passes through them are called phase objects [1]. In this way, it is possible to observe living cells and organisms with adequate contrast and resolution. DIC produces relief-like images where structures and features of the specimen seem to cast a shadow. They have no halo artifacts and relatively thick specimens can also be imaged due to the possibility of optical sectioning.

The technique of DIC microscopy was first invented by Francis Smith in 1947, but then further developed and made into a practical microscopy method by Georges Nomarski in 1952 [2].

For DIC microscopy, only polarized light is used to illuminate the specimen. The polarized light is dispersed into two distinct light rays which are in orthogonal planes of polarization. These two light rays are extremely near to each other. As they experience refraction or scattering in different ways within the specimen, then 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 with wavelength differences between 1/200 of a wavelength (or even 1/1000 of a wavelength when using a camera) and a whole wavelength can be made visible.

Figure 1: Neurons imaged with brightfield and DIC contrast.

Light wave’s oscillations

For non-polarized light, the light waves oscillate in all directions with respect to their axis of propagation. However, in linearly polarized light, all wave oscillations have the same angle or plane of polarization. Circular polarization is another possible type of polarization. The light-wave 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 material or 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.

Figure 2: Examples of polarized light: Composition of a linearly, circularly, or elliptically polarized wave (black) which results from a superposition of linearly polarized components (red and gray).

Optical path in a DIC microscope

In a DIC microscope, polarized light is produced via a polarizer placed 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 which is better known as a Wollaston prism. The Wollaston prism can be found in the plane of the condenser. A standard Wollaston prism is made from two wedges of a birefringent material that are cemented together on their base and have optical axes parallel to the outer surface and perpendicular to each other.

Figure 3: In the first part of the Wollaston prism, 45° polarized light is separated into two perpendicular polarized rays with an angle of 0° and 90°, respectively (left). The second part of the prism subsequently changes their dispersion according to the polarization (right). Both rays become parallel after passing through the condenser.

Polarized light passing through this prism is sheared into two closely spaced waves with different angles of deflection: the ordinary and extraordinary wave. They have perpendicular planes of polarization and for these two waves the medium behaves as if it has effectively a single refractive index. The shearing 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 in parallel with a lateral displacement. If these two light waves interact with specimen parts having 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 traverse the specimen, they are brought back together via another Wollaston prism. Now they interfere with each other, forming elliptically polarized light which then 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 difference leads to an interference plane which lies outside the prism. As a result, the prism can be located outside the objective’s aperture plane, making DIC easier to use.

For DIC microscopy, the phase shift differences of neighboring object points contribute to the image formation. Details of the specimen, which have a gradient in terms of their refractive indices or thicknesses, are more clearly visualized. As the light beams are polarized perpendicularly to each other, a difference in the images can be seen by rotating the microscope stage.

It is important to remember not to use plastic substrates, slides, or coverslips with specimens when performing DIC microscopy, because many polymers have a depolarizing effect on light and often distort the contrast.

Figure 4: Unpolarized light passes through a polarizing filter and is then polarized at 45°. After passing through the Wollaston prism, the light is separated into perpendicular polarized components, one with 0° and the other 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 constructively or destructively interfere. The separated rays experience different optical path lengths, as there might be differences in thickness or refractive index at the points where 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 rays are recombined into one polarized at 135°. Depending to differences in the optical path length, interference of the two rays now produces brightening or darkening of specimen areas, making structures appear which are otherwise hardly visible with brightfield illumination. Finally, directly transmitted light, which did not experience any phase shift, is removed by the 135° polarizing filter (also called analyzer).