DIFFERENTIAL INTERFERENCE CONTRAST (DIC)

From Ruzin, 1999. Plant Microtechnique and Microscopy

Differential Interference Contrast is another method of deriving contrast in an unstained specimen from differences in index of refraction of specimen components. As with Phase Contrast, DIC transforms the phase shift of light, induced by the specimen refractive index, into detectable amplitude differences. An advantage of interference-derived contrast is that an object will appear bright against a dark background but without the diffraction halo associated with phase contrast. However, because DIC utilizes optical path differences within the specimen (i.e.: product of refractive index and geometric path length) to generate contrast the three-dimensional appearance may not represent reality. In other words the 3-D relief of DIC imaged specimens is an optical rather than a geometric relief.

Differential interference contrast microscopes are actually microscope interferometers in that they generate contrast within the specimen by exploiting phase differences between a specimen light ray and a reference ray. In interferometry, a single ray of light is split into two rays, one traversing and the other missing the specimen but interacting with the background (Figure 1-4). The rays are then recombined at the image plane, where wave interference may occur. Image contrast can be modified by altering the phase difference between the reference and specimen rays.

In differential interference contrast microscopy, the sample and reference rays are created after the ray traverses, and is phase-distorted by, the sample (Figure 1-5). The phased ray then is split into two equally phased, but spatially separated ("sheared") rays by the action of a Wollaston prism. Finally, the ray-pairs pass through a polarizing filter where they are vibrationally recombined and interact at the image plane. It is the wave interference of these two rays that creates contrast.

Figure 1-4 Principle of an interferometer.

A light beam (S) is split into two beams at M where one beam passes through the specimen (Spec) and the other ("reference beam", Ref) passes through the background. Subsequently the two beams are recombined at N where wave interference occurs resulting in the image beam (S').

As with Phase contrast, differential interference contrast relies on the insertion and alignment of additional optical components (Figure 1-5, C): a polarizing filter placed between the light source and condenser to output plane polarized light and a two-layered Nomarski-modified Wollaston prism that separates individual rays of light into ray pairs (Figure 1-5, A; E, O) that are in phase but spatially separated and vibrating at 90° apart. The lower Wollaston prism (W1, Figure 1-5, C) is built into the condenser complex of the microscope. The upper prism (W2, Figure 1-5, C) is placed above the objective and is laterally adjustable. In Smith-type DIC, a conventional Wollaston prism is placed in the back focal plane of the objective. In Nomarski DIC, a modified Wollaston prism is placed above the objective in a position slightly beyond the objective back focal plane. Optically above the top Wollaston prism is placed another polarizing filter (Analyzer; Ana, Figure 1-5, C) that recombines the vibrational planes of the ray-pairs, allowing wave interference to occur.

How DIC works

Figure 1-5 Nomarski Differential Interference Contrast microscopy.

Refer to Figure 1-5, B for the following discussion of differential interference contrast microscopy.

  1. A plane-polarized wavefront (Ep) created by the action of the condenser and polarizer (Pol) passes through the specimen.
  2. Local optical path differences within the specimen (Spec), caused by differences in index of refraction, create a phase distorted wavefront (E). Phase distortion is represented by the displaced portion of the wavefront.
  3. Wavefront E is collected by the objective lens (Obj) and is "sheared" into two offset wavefronts (Eo and Ee) by the action of the upper Wollaston prism (W2). Due to the optical path difference between the ray-pairs as they traverse different portions of the upper prism (cf. Figure 1-5, A, i and ii) the specimen image is duplicated both laterally and longitudinally (D). No interaction yet occurs between the wavefronts as they are vibrating at right angles to each other.
  4. The wavefronts then pass through the Analyzer (Ana) where they are recombined into one vibrational plane. It is at this point where constructive or destructive wave interference occurs. This causes optical path differences within the sample to be manifested as light or dark areas in the image.
  5. Where no interaction occurs between ray-pairs, interference of equally phased rays produces a uniform gray background.

Thus it is the interference of two difference images that results in the contrast of the image visualized. This imaging method is therefore appropriately called Differential Interference Contrast.

A: Birefringence of the Wollaston prism separates a plane-polarized light ray into two spatially separated rays vibrating 90° apart. The position of the impinging ray relative to the prism determines the phase relationship between the resulting ray pairs (EO: separated e-ray and o-ray). Difference images d1 and d2 represent possible phase relationships resulting from positional changes in the upper Wollaston prism.

B: Creation of a duplicated image (Eo and Ee) by a birefringent Wollaston prism. Polarized wave (Ep) passing through the specimen creates a plane wave (E) that is separated into two components by the upper Wollaston prism (W2). After passing through the Analyzer (Ana), a differential image is created by wave interference of the two duplicated images having a wave phase difference of Æ. Pol: lower polarizer; Obj: objective lens; Spec: specimen

C: The arrangement of components of the Nomarski interference microscopes (C). Pol: polarizer; W1: lower Nomarski-modified Wollaston prism; fc: Condenser focal plane; Cond: condenser lens; Spec: specimen; Obj: objective lens; f0: back focal plane of the objective lens; W2: Upper Nomarski-modified Wollaston prism; Ana: upper polarizer (Analyzer). Double-headed arrow shows the possible lateral movement of W2.

The function of the lower Wollaston prism

DIC originally required a slit condenser to illuminate the sample with a vertical plane of coherent light. However, this configuration severely constrains the amount of light that can be used to illuminate the sample. The addition of a Nomarski-modified Wollaston prism below the condenser (Figure 1-5C, W1) allows the use of a full condenser aperture. This lower prism is also sometimes called a DIC Compensator because it compensates for optical path differences between all possible off-axis rays relative to on-axis rays. That is, phase differences of all light rays emanating from the full width of the condenser aperture are compensated for by the prism. Using a Wollaston prism in this way allows the entire condenser aperture to be utilized for specimen illumination, which results in an overall brighter image.

Setting up a DIC microscope

Functionally, setting up DIC is easy. The components for DIC are mirror image pairs: a polarizer and Wollaston prism below the specimen and another Wollaston prism and polarizer above the specimen. All four components must be in place for Nomarski DIC to function. Contrast is adjusted by changing the lateral position of the upper Wollaston prism thus altering the phase difference between ray-pairs. Of course, the microscope should be adjusted for Köhler illumination before fixing the DIC components in place.