Differential interference contrast microscopy
A relatively complex optical system produces an image
Micrasterias furcata imaged in transmitted DIC microscopy.
Laser-induced optical damage in LiNbO3 under 150× Nomarski
microscopy.
Differential interference contrast (DIC) microscopy,
also known as Nomarski interference contrast (NIC)
or Nomarski microscopy, is an optical microscopy
technique used to enhance the contrast in unstained,
transparent samples. DIC works on the principle of
interferometry to gain information about the optical path
length of the sample, to see otherwise invisible features.
1
with the object appearing black to white on a grey back-
ground. This image is similar to that obtained by phase
contrast microscopy but without the bright diffraction
halo. The technique was developed by polish physicist
Georges Nomarski in 1952.[1]
DIC works by separating a polarized light source into
two orthogonally polarized mutually coherent parts which
are spatially displaced (sheared) at the sample plane,
and recombined before observation. The interference of
the two parts at recombination is sensitive to their opti-
cal path difference (i.e. the product of refractive index
and geometric path length). Adding an adjustable offset
phase determining the interference at zero optical path
difference in the sample, the contrast is proportional to
the path length gradient along the shear direction, giving
the appearance of a three-dimensional physical relief cor-
responding to the variation of optical density of the sam-
ple, emphasising lines and edges though not providing a
topographically accurate image.
1 The light path
1. Unpolarised light enters the microscope and is polarised
at 45°.
Polarised light is required for the technique to
work.
2. The polarised light enters the first Nomarski-modified
Wollaston prism and is separated into two rays polarised
at 90° to each other, the sampling and reference rays.
Wollaston prisms are a type of prism made of
two layers of a crystalline substance, such as
quartz, which, due to the variation of refrac-
tive index depending on the polarisation of the
light, splits the light according to its polarisa-
tion. The Nomarski prism causes the two rays
to come to a focal point outside the body of
the prism, and so allows greater flexibility when
setting up the microscope, as the prism can be
actively focused.
3. The two rays are focused by the condenser for passage
through the sample. These two rays are focused so they
will pass through two adjacent points in the sample, around
0.2 μm apart.
2 2 IMAGE

The sample is effectively illuminated by two in phase is due to the difference in optical path
coherent light sources, one with 0° polarisation length, this recombination of light causes “op-
and the other with 90° polarisation. These two tical differentiation" of the optical path length,
illuminations are, however, not quite aligned, generating the image seen.
with one lying slightly offset with respect to the
other.
2 Image

The route of light through a DIC microscope. The two light beams
should be parallel between condenser and objective

4. The rays travel through adjacent areas of the sample,

separated by the shear. The separation is normally similar
to the resolution of the microscope. They will experience
different optical path lengths where the areas differ in re-
fractive index or thickness. This causes a change in phase
of one ray relative to the other due to the delay experienced
by the wave in the more optically dense material.

The passage of many pairs of rays through pairs

of adjacent points in the sample (and their ab-
sorbance, refraction and scattering by the sam-
ple) means an image of the sample will now
be carried by both the 0° and 90° polarised
light. These, if looked at individually, would be
bright field images of the sample, slightly off-
set from each other. The light also carries in-
formation about the image invisible to the hu-
man eye, the phase of the light. This is vital
later. The different polarisations prevent inter-
ference between these two images at this point.
An illustration of the process of image production in a DIC mi-
croscope.
5. The rays travel through the objective lens and are fo-
cused for the second Nomarski-modified Wollaston prism. The image has the appearance of a three-dimensional ob-
ject under very oblique illumination, causing strong light
6. The second prism recombines the two rays into one
and dark shadows on the corresponding faces. The direc-
polarised at 135°. The combination of the rays leads to
tion of apparent illumination is defined by the orientation
interference, brightening or darkening the image at that
of the Wollaston prisms.
point according to the optical path difference.
As explained above, the image is generated from two
This prism overlays the two bright field im- identical bright field images being overlaid slightly offset
ages and aligns their polarisations so they can from each other (typically around 0.2 μm), and the sub-
interfere. However, the images do not quite sequent interference due to phase difference converting
line up because of the offset in illumination changes in phase (and so optical path length) to a visible
- this means that instead of interference oc- change in darkness. This interference may be either con-
curring between 2 rays of light that passed structive or destructive, giving rise to the characteristic
through the same point in the specimen, inter- appearance of three dimensions.
ference occurs between rays of light that went The typical phase difference giving rise to the interfer-
through adjacent points which therefore have a ence is very small, very rarely being larger than 90° (a
slightly different phase. Because the difference quarter of the wavelength). This is due to the similarity of
 3

refractive index of most samples and the media they are

in: for example, a cell in water only has a refractive index
difference of around 0.05. This small phase difference
is important for the correct function of DIC, since if the
phase difference at the joint between two substances is
too large then the phase difference could reach 180° (half
a wavelength), resulting in complete destructive interfer-
ence and an anomalous dark region; if the phase differ-
ence reached 360° (a full wavelength), it would produce
complete constructive interference, creating an anoma-
lous bright region.
The image can be approximated (neglecting refraction
and absorption due to the sample and the resolution limit
of beam separation) as the differential of optical path
length with respect to position across the sample along Partially developed photoresist via Nomarski DIC
the shear, and so the differential of the refractive index
(optical density) of the sample.

DIC images with different offset phases φ0 .

The contrast can be adjusted using the offset phase, ei-

ther by translating the objective Nomarski prism, or by a
lambda/4 waveplate between polarizer and the condenser
Normarski prism (De-Senarmont Compensation). The Aluminum-Silicon alloying pit made visible via Nomarski DIC
resulting contrast is going from dark-field for zero phase
offset (intensity proportional to the square of the shear
differential), to the typical relief seen for phase of ~5–90
degrees, to optical staining at 360 degrees, where the ex-
tinguished wavelength shifts with the phase differential.

3 Applications

Partially etched silicon dioxide via Nomarski DIC

sis of planar silicon semiconductor processing. The thin

Orientation specific imaging of a transparent cuboid in DIC.
DIC is used for imaging live and unstained biological
samples, such as a smear from a tissue culture or indi-
vidual water borne single-celled organisms. Its resolution
and clarity in conditions such as this are unrivaled among
standard optical microscopy techniques.
One non-biological area where DIC is used is in the analy-
(typically 100–1000 nm) films in silicon processing are
often mostly transparent to visible light (e.g., silicon diox-
ide, silicon nitride and polycrystalline silicon), and de-
fects in them or contamination lying on top of them be-
come more visible. This also enables the determination
of whether a feature is a pit in the substrate material or
a blob of foreign material on top. Etched crystalline fea-
tures gain a particularly striking appearance under DIC.
4 6 EXTERNAL LINKS

Image quality, when used under suitable conditions, is

outstanding in resolution and almost entirely free of ar-
tifacts unlike phase contrast. However analysis of DIC
images must always take into account the orientation of
the Wollaston prisms and the apparent lighting direction,
as features parallel to this will not be visible. This is, how-
ever, easily overcome by simply rotating the sample and
observing changes in the image.

4 See also
• Classical interference microscopy
• Nanovid microscopy

5 References
• Murphy, D., Differential interference contrast
(DIC) microscopy and modulation contrast mi-
croscopy, in Fundamentals of Light Microscopy and
Digital Imaging, Wiley-Liss, New York, pp. 153–
168 (2001).
• Salmon, E. and Tran, P., High-resolution video-
enhanced differential interference contrast (VE-
DIC) light microscope., Video Microscopy, Sluder,
G. and Wolf, D. (eds), Academic Press, New York,
pp. 153–184 (1998).

• Differential Interference Contrast — references

6 External links
• Molecular Expressions:
• Differential Interference Contrast Primer

• Differential Interference Contrast

[1] Lang, Walter (1968). “Nomarski differential

interference-contrast microscopy” (PDF). ZEISS In-
formation. 70: 114–120. Retrieved 31 August 2016.
 5

7 Text and image sources, contributors, and licenses

7.1 Text
• Differential interference contrast microscopy Source: https://en.wikipedia.org/wiki/Differential_interference_contrast_microscopy?
oldid=737050540 Contributors: Chinasaur, St3vo, Katana, TenOfAllTrades, Woohookitty, Srleffler, Spacepotato, Peter G Werner,
Apokryltaros, Mysid, Zephyris, Thumperward, DroEsperanto, EdgeOfEpsilon, Grothmag, CmdrObot, Frozenport, Bobblehead, Seaphoto,
Pvosta, IanHarvey, Daidaiking, Dietzel65, CommonsDelinker, Richwil, Biscuittin, Richstraka, Twinsday, Mild Bill Hiccup, SchreiberBike,
Matthew Desjardins, Inductionheating, Ncross35, Addbot, Innv, MrOllie, Luckas-bot, Deltafunction~enwiki, AnomieBOT, Tryptofish,
TechBot, J04n, Wolftrans, Televiisor, Pieter-Jan Goossens, Kleopatra, ClueBot NG, Bomos, Leica Microsystems, Amadori Heyns, Li-
braryStudent24, Shibbolethink, L0st H0r!z0ns, Jason Yager and Anonymous: 28

7.2 Images
• File:1-1-1_Pits_from_Aluminum_Alloying.jpg Source: https://upload.wikimedia.org/wikipedia/commons/3/33/1-1-1_Pits_from_
Aluminum_Alloying.jpg License: CC BY-SA 3.0 Contributors: Own work Original artist: Richstraka
• File:Al_photoresist_pattern_developed_via_Nomarski_DIC.jpg Source: https://upload.wikimedia.org/wikipedia/commons/6/67/Al_
photoresist_pattern_developed_via_Nomarski_DIC.jpg License: CC BY-SA 3.0 Contributors: Own work Original artist: Richstraka
• File:DIC_Example.png Source: https://upload.wikimedia.org/wikipedia/commons/3/3f/DIC_Example.png License: CC-BY-SA-3.0
Contributors: en:Image:DIC Example.png Original artist: Richard Wheeler (Zephyris)
• File:DIC_Light_Path.png Source: https://upload.wikimedia.org/wikipedia/commons/4/46/DIC_Light_Path.png License: CC-BY-SA-
3.0 Contributors: en:Image:DIC Light Path.png Original artist: Richard Wheeler (Zephyris)
• File:DIC_Limitation_Example.png Source: https://upload.wikimedia.org/wikipedia/commons/3/3f/DIC_Limitation_Example.png Li-
cense: CC-BY-SA-3.0 Contributors: en:Image:DIC Limitation Example.png Original artist: Richard Wheeler (Zephyris)
• File:DIC_Phase.jpg Source: https://upload.wikimedia.org/wikipedia/commons/9/9f/DIC_Phase.jpg License: CC BY-SA 3.0 Contribu-
tors: Own work Original artist: Wolftrans
• File:Laser-Induced_Optical_Damage_in_LiNbO3.jpg Source: https://upload.wikimedia.org/wikipedia/commons/4/4d/
Laser-Induced_Optical_Damage_in_LiNbO3.jpg License: CC BY-SA 4.0 Contributors: Own work Original artist: Jason Yager
• File:Loupe-binoculaire-p1030891.jpg Source: https://upload.wikimedia.org/wikipedia/commons/c/c3/Loupe-binoculaire-p1030891.
jpg License: CC BY-SA 2.0 fr Contributors: Own work Original artist: Rama
• File:Micrasterias_radiata.jpg Source: https://upload.wikimedia.org/wikipedia/commons/4/47/Micrasterias_radiata.jpg License: CC
BY-SA 2.5 Contributors: Own work Original artist: ja:User:NEON / User:NEON_ja
• File:Partially_etched_silicon_dioxide_via_Nomarski_DIC.jpg Source: https://upload.wikimedia.org/wikipedia/commons/b/b4/
Partially_etched_silicon_dioxide_via_Nomarski_DIC.jpg License: CC BY-SA 3.0 Contributors: Own work Original artist: Richstraka

7.3 Content license

• Creative Commons Attribution-Share Alike 3.0