Differential interference contrast (DIC) microscopy explained

If you’re looking for a microscopy technique and are feeling flush, then DIC might be the answer. 

What is DIC?

DIC microscopy is an advanced optical technique for enhancing contrast in transparent samples.

It’s particularly useful for the study of living and unstained specimens. It also has applications in fluorescence microscopy.

How it works

As the name implies, DIC uses interference to generate additional contrast in an image, letting you see ordinarily invisible details.

Light is split into 2 separate rays and then transmitted through a sample. Afterwards, both rays are recombined back into a single ray.

Passing through the sample causes the rays to shift their phase, so when they’re reunited the phase differences between the two now-distinct rays cause interference, which is then translated into a visual difference.

Essentially, DIC sends 2 parallel rays of light through a sample, then uses the differences between them to measure the gradient of the optical path. The parts of the sample that produced a steeper gradient because of their thickness or higher refractive index show up darker in the image.

This produces high-quality images that look almost 3D – they appear to have shadows and a depth that can occasionally be misleading.

Despite this, DIC images are high resolution and have excellent contrast, especially compared to other optical techniques, making it a popular and useful method for inspecting transparent samples, such as microorganisms or tissue cells.

The mechanics

DIC works by using a combination of polarized light and special prisms, known as Wollaston or Nomarski prisms.

Step 1

The light from the illuminator passes through a polarizing filter.

Light waves normally vibrate in random directions. Polarizing filters are like a set of bars that only allow the light that is oriented in a particular direction to get through. In this case, only light vibrating at a 45-degree angle can get through.

Step 2

The now-polarized light enters the Wollaston or Nomarski prism.

These prisms split the light ray into 2 separate rays with different polarities. One is polarized at zero degrees, while the other is now perpendicular at 90 degrees. The 2 rays are still very close to each other but won’t interfere with one another because of their different polarities.

Step 3

The 2 separate rays then pass through the sample.

Where the rays encounter parts of the sample, they are scattered or refracted by the sample’s thickness or different refractive index. Each ray will experience a different journey, so there will be a difference in phase between them.

Step 4

Both rays then pass through a second Wollaston or Nomarski prism, where they are merged back into a single ray.

This time, rather than giving them different polarities, the prism brings both light rays together at the same polarity – 135 degrees.

Because they’re now at the same polarity, the phase differences between them cause interference. This difference is converted into amplitude shifts – showing brighter or darker areas in the image.