![Laser-Induced Optical Damage in LiNbO3](https://upload.wikimedia.org/wikipedia/commons/4/4d/Laser-Induced_Optical_Damage_in_LiNbO3.jpg)
Differential interference contrast (DIC) microscopy, also known as Nomarski interference contrast (NIC) or Nomarski microscopy, is an
optical microscopy
Optics is the branch of physics that studies the behaviour and properties of light, including its interactions with matter and the construction of instruments that use or detect it. Optics usually describes the behaviour of visible, ultraviole ...
technique used to enhance the
contrast in unstained, transparent
samples. DIC works on the principle of
interferometry
Interferometry is a technique which uses the ''interference'' of superimposed waves to extract information. Interferometry typically uses electromagnetic waves and is an important investigative technique in the fields of astronomy, fiber opt ...
to gain information about the
optical path length In optics, optical path length (OPL, denoted ''Λ'' in equations), also known as optical length or optical distance, is the product of the geometric length of the optical path followed by light and the refractive index of homogeneous medium through ...
of the sample, to see otherwise invisible features. A relatively complex optical system produces an image with the object appearing black to white on a grey background. This image is similar to that obtained by
phase contrast microscopy
__NOTOC__
Phase-contrast microscopy (PCM) is an optical microscopy technique that converts phase shifts in light passing through a transparent specimen to brightness changes in the image. Phase shifts themselves are invisible, but become visible ...
but without the bright diffraction halo. The technique was developed by Polish physicist
Georges Nomarski Georges (Jerzy) Nomarski (January 6, 1919 – 1997) was a Polish physicist and optics theoretician. Creator of differential interference contrast (DIC) microscopy, the method is widely used to study live biological specimens and unstained tissues an ...
in 1952.
DIC works by separating a
polarized light source into two
orthogonally
In mathematics, orthogonality is the generalization of the geometric notion of ''perpendicularity''.
By extension, orthogonality is also used to refer to the separation of specific features of a system. The term also has specialized meanings in ...
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 optical path difference (i.e. the product of
refractive index
In optics, the refractive index (or refraction index) of an optical medium is a dimensionless number that gives the indication of the light bending ability of that medium.
The refractive index determines how much the path of light is bent, or ...
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 corresponding to the variation of optical density of the sample, emphasising lines and edges though not providing a topographically accurate image.
The light path
1. ''Unpolarised light enters the
microscope
A microscope () is a laboratory instrument used to examine objects that are too small to be seen by the naked eye. Microscopy is the science of investigating small objects and structures using a microscope. Microscopic means being invisibl ...
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
A Wollaston prism is an optical device, invented by William Hyde Wollaston, that manipulates polarized light. It separates light into two separate linearly polarized outgoing beams with orthogonal polarization. The two beams will be polarized a ...
and is separated into two
ray
Ray may refer to:
Fish
* Ray (fish), any cartilaginous fish of the superorder Batoidea
* Ray (fish fin anatomy), a bony or horny spine on a fin
Science and mathematics
* Ray (geometry), half of a line proceeding from an initial point
* Ray (g ...
s 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 refractive index depending on the polarisation of the light, splits the light according to its polarisation. The
Nomarski prism
A Nomarski prism is a modification of the Wollaston prism that is used in differential interference contrast microscopy. It is named after its inventor, Polish and naturalized-French physicist Georges Nomarski. Like the Wollaston prism, the Nomar ...
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.''
: The sample is effectively illuminated by two
coherent
Coherence, coherency, or coherent may refer to the following:
Physics
* Coherence (physics), an ideal property of waves that enables stationary (i.e. temporally and spatially constant) interference
* Coherence (units of measurement), a deri ...
light sources, one with 0° polarisation and the other with 90° polarisation. These two illuminations are, however, not quite aligned, with one lying slightly offset with respect to the other.
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 refractive index or thickness. This causes a change in
phase
Phase or phases may refer to:
Science
*State of matter, or phase, one of the distinct forms in which matter can exist
*Phase (matter), a region of space throughout which all physical properties are essentially uniform
* Phase space, a mathematic ...
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 absorbance,
refraction
In physics, refraction is the redirection of a wave as it passes from one medium to another. The redirection can be caused by the wave's change in speed or by a change in the medium. Refraction of light is the most commonly observed phenomeno ...
and scattering by the sample) 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 offset from each other. The light also carries information about the image invisible to the human eye, the phase of the light. This is vital later. The different polarisations prevent interference between these two images at this point.
5. ''The rays travel through the
objective lens
In optical engineering, the objective is the optical element that gathers light from the object being observed and Focus (optics), focuses the ray (optics), light rays to produce a real image. Objectives can be a single Lens (optics), lens or mirr ...
and are focused for the second Nomarski-modified Wollaston prism.''
6. ''The second
prism
Prism usually refers to:
* Prism (optics), a transparent optical component with flat surfaces that refract light
* Prism (geometry), a kind of polyhedron
Prism may also refer to:
Science and mathematics
* Prism (geology), a type of sedimentary ...
recombines the two rays into one polarised at 135°. The combination of the rays leads to
interference
Interference is the act of interfering, invading, or poaching. Interference may also refer to:
Communications
* Interference (communication), anything which alters, modifies, or disrupts a message
* Adjacent-channel interference, caused by extra ...
, brightening or darkening the image at that point according to the optical path difference.''
: This prism overlays the two bright field images and aligns their polarisations so they can interfere. However, the images do not quite line up because of the offset in illumination – this means that instead of interference occurring between 2 rays of light that passed through the same point in the specimen, interference occurs between rays of light that went through ''adjacent'' points which therefore have a slightly different phase. Because the difference in phase is due to the difference in optical path length, this recombination of light causes "optical
differentiation" of the optical path length, generating the image seen.
Image
The image has the appearance of a three-dimensional object under very oblique illumination, causing strong light and dark shadows on the corresponding faces. The direction of apparent illumination is defined by the orientation of the Wollaston prisms.
As explained above, the image is generated from two identical bright field images being overlaid slightly offset from each other (typically around 0.2 μm), and the subsequent interference due to phase difference converting changes in phase (and so optical path length) to a visible change in darkness. This interference may be either constructive or destructive, giving rise to the characteristic appearance of three dimensions.
The typical phase difference giving rise to the interference is very small, very rarely being larger than 90° (a quarter of the wavelength). This is due to the similarity of 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 interference and an anomalous dark region; if the phase difference reached 360° (a full wavelength), it would produce complete constructive interference, creating an anomalous 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 the shear, and so the differential of the refractive index (optical density) of the sample.
The contrast can be adjusted using the offset phase, either by translating the objective Nomarski prism, or by a lambda/4 waveplate between polarizer and the condenser Normarski prism (De-Senarmont Compensation). The 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 extinguished wavelength shifts with the phase differential.
When sequentially shifted images are collated, the phase-shift introduced by the object can be decoupled from unwanted non-interferometric artifacts, which typically results in an improvement in contrast, especially in turbid samples.
Applications
![Al_photoresist_pattern_developed_via_Nomarski_DIC](https://upload.wikimedia.org/wikipedia/commons/6/67/Al_photoresist_pattern_developed_via_Nomarski_DIC.jpg)
DIC is used for imaging live and unstained biological samples, such as a smear from a tissue culture or individual water borne single-celled organisms. Its resolution and clarity in conditions such as this are unrivaled among standard optical microscopy techniques.
![Partially_etched_silicon_dioxide_via_Nomarski_DIC](https://upload.wikimedia.org/wikipedia/commons/b/b4/Partially_etched_silicon_dioxide_via_Nomarski_DIC.jpg)
One non-biological area where DIC is used is in the analysis of planar silicon semiconductor processing. The thin (typically 100–1000 nm) films in silicon processing are often mostly transparent to visible light (e.g., silicon dioxide, silicon nitride and polycrystalline silicon), and defects in them or contamination lying on top of them become 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 features gain a particularly striking appearance under DIC.
Image quality, when used under suitable conditions, is outstanding in resolution and almost entirely free of artifacts 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, however, easily overcome by simply rotating the sample and observing changes in the image.
See also
*
Classical interference microscopy Classical interference microscopy, also called quantitative interference microscopy, uses two separate light beams with much greater lateral separation than that used in phase contrast microscopy or in differential interference microscopy (DIC).
I ...
*
Nanovid microscopy
References
* Murphy, D., Differential interference contrast (DIC) microscopy and modulation contrast microscopy, 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
External links
Reflected Light DIC MicroscopyA guide to Differential Interference Contrast (DIC)
{{Optical microscopy
Interferometry
Laboratory equipment
Optical microscopy