What DIC is
Differential interference contrast (DIC) microscopy is a technique for making transparent, unstained specimens visible with high contrast. It produces a distinctive image that looks shaded, as if each cell were lit from one side, giving a strong impression of relief. It’s also called Nomarski microscopy or Nomarski interference contrast, after Georges Nomarski, who developed the modified prism design widely used in the 1950s.
Like phase contrast, DIC detects tiny differences in how light is delayed as it passes through a specimen. It does this in a different way, though, and the image looks quite different.
How DIC works, step by step
A transmitted-light DIC microscope adds four optical parts to a standard microscope:
- Polarizer (below the condenser). It makes the light plane-polarized.
- First prism (in or below the condenser). It’s a Wollaston prism, or a modified Wollaston prism known as a Nomarski prism. It splits each ray of light into two rays polarized at right angles to each other and separated sideways by a very small distance, called the shear. The shear is typically smaller than the resolution limit of the objective.
- Specimen. The two closely spaced rays pass through neighboring points of the specimen. If those points differ in thickness or refractive index, one ray is delayed slightly more than the other.
- Second prism (behind the objective, often in a slider). It recombines the two rays.
- Analyzer (above the objective). A second polarizer, crossed with the first. It lets the recombined rays interfere, converting their path difference into a change in brightness.
Where neighboring points delay the light equally, as in a flat background or a uniform region, nothing much changes. Where the delay changes from point to point, as at an edge or a slope in optical thickness, the two rays come out of step and the brightness changes. DIC is therefore sensitive to the gradient (rate of change) of optical path. That’s the “differential” in its name.
Sliding the second prism sideways adds a controllable offset, called the bias. It sets the background to gray and makes one side of each feature bright and the other side dark. That’s what produces the shadow-cast look.
What a DIC image looks like (and what the “3D” really means)
DIC images look strongly three-dimensional. Cells look like embossed shapes on a gray background, with highlights on one side and shadows on the other along the shear direction.
That relief is not true topography. It reflects changes in optical path, which combine thickness and refractive index, along one direction. A feature that sticks up and one that has a higher refractive index can look the same, and edges running parallel to the shear direction show little contrast. Rotating the specimen, or the prisms on some microscopes, changes which edges are emphasized.
Advantages
- No staining needed, so living cells can be observed.
- No bright halos of the kind seen in phase contrast, so edges look cleaner.
- Uses the full aperture. DIC works with the condenser and objective at or near full numerical aperture, so it keeps resolution high.
- Shallow depth of field (“optical sectioning”). Because it works at high aperture, DIC shows a thin slice of the specimen in focus, with relatively little interference from structures above and below. That makes it useful for thicker specimens such as embryos and tissue slices.
- Adjustable contrast. You can tune the bias for each specimen.
- Combines well with fluorescence. DIC is often used alongside fluorescence microscopy to show the context around labeled structures. Infrared DIC is also widely used to see neurons deep in brain slices for electrophysiology.
Disadvantages
- Cost. Prisms, polarizers and DIC-rated objectives are expensive, and each objective generally needs a matching prism.
- Birefringent materials interfere. Because DIC relies on polarized light, anything birefringent in the path disturbs the image. Standard plastic culture dishes are a well-known problem, which is one reason labs use glass-bottomed dishes or Hoffman modulation contrast with plastic.
- Orientation dependence. Contrast depends on the direction of features relative to the shear.
- The relief can mislead. As noted above, the “3D” appearance isn’t a reliable map of height.
- More setup. Polarizer, analyzer, prisms and bias all need adjusting, on top of normal Köhler illumination.
- Strongly birefringent specimens such as some crystals produce colorful, confusing images. Those are better studied with polarized light microscopy.
DIC vs phase contrast
| DIC | Phase contrast | |
|---|---|---|
| Responds to | Gradient of optical path (changes from point to point) | Optical path differences relative to background |
| Image | Shadow-cast relief on gray | Darker or brighter features on gray, with halos |
| Halos | No | Yes, around edges |
| Aperture | Full or near-full; high resolution | Restricted by the annulus |
| Thick specimens | Works reasonably well, with optical sectioning | Poor |
| Plastic dishes | Problematic | Fine |
| Cost | High | Moderate |
| Ease of use | More adjustments | Simpler once annuli are centered |
Phase contrast is often the practical choice for routine viewing of thin cell layers and is better with plastic vessels. DIC gives cleaner edges and better results on thicker specimens, at a higher price.
Reflected-light DIC
DIC also works in reflected light. On metallurgical microscopes, a single prism is used in the illumination and imaging path, and the technique shows very small height differences on polished surfaces, such as grain boundaries, polishing marks and defects on wafers and coatings. See transmitted vs reflected light microscopes for how reflected illumination works.
DIC for amateurs
New DIC equipment is usually beyond a hobby budget, but there are options:
- Used equipment. Older research microscopes with DIC components sometimes appear secondhand (see our used microscope checklist). Check that the condenser prisms, objective prisms and objectives all belong to the same system and match each other. Parts from different systems usually don’t work together.
- Oblique illumination. You can get a relief effect that looks somewhat similar by lighting the specimen from one side. Partly blocking one side of the light path below the condenser with a card, or offsetting the condenser’s aperture diaphragm if your microscope allows it, produces shadow-cast images of diatoms, protozoa and cells at almost no cost. It doesn’t match DIC’s optical sectioning or full-aperture resolution, but it’s a good way to try the look.
- Other low-cost methods. A homemade darkfield stop and simple polarizing filters are also cheap ways to explore contrast on transparent specimens.
For the bigger picture of where DIC sits among contrast methods, see contrast in microscopy.