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Encyclopedia > Differential interference contrast microscopy

Differential interference contrast microscopy (DIC), also known as Nomarski Interference Contrast (NIC) or Nomarski microscopy, is an optical microscopy illumination technique used to enhance the contrast in unstained samples. DIC works on the principle of inferometry to gain information about the optical density of the sample, to see otherwise invisible features. It is a relatively complex lighting scheme that produces an image with the object appearing black to white on a grey background, similar to phase contrast microscopy, but without the bright diffraction halo. A microscope (Greek: micron = small and scopos = aim) is an instrument for viewing objects that are too small to be seen by the naked or unaided eye. ... Illumination is either Illumination as the practice of living in Love and Light lighting — supplying light to an area Enlightenment (Satori), see also Illuminati the art of decorating letters or pages with ink and embossing techniques. ... Look up Contrast in Wiktionary, the free dictionary. ... In general, a sample is a part of the total, such as one individual or a set of individuals from a population (of people or things), a small piece or amount of something larger, a number of function values of a function, or part of a song. ... Optical density is the absorbance of an optical element for a given wavelength λ per unit distance: Where: = the distance that light travels through the sample (i. ... A phase contrast microscope is a microscope that does not require staining to view the slide. ...


DIC works by separating a polarised light source into two beams which take slightly different paths through the sample. These take different optical path lengths (ie. product of refractive index and geometric path length), so when they are recombined interfere. This gives the appearance of physical relief from the variation of optical density of the sample. This article treats polarization in electrodynamics. ... The refractive index (or index of refraction) of a material is the factor by which the phase velocity of electromagnetic radiation is slowed in that material, relative to its velocity in a vacuum. ...

Contents

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The light path

The components of the basic differential interference contrast microscope setup.
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The components of the basic differential interference contrast microscope setup.

1. Unpolarised light enters the microscope and is polarised at 45°. A microscope (Greek: micron = small and scopos = aim) is an instrument for viewing objects that are too small to be seen by the naked or unaided eye. ...

Polarised light is required because of the way this technique depends on the interference of polarised light - un-polarised light initially entering the sample could disrupt this.

2. The polarised light enters the first Nomarski-modified Wollaston prism and is separated into two rays polarised at 90° to each other, the ordinary and extraordinary rays. The Wollaston prism is an optical device, invented by William Hyde Wollaston, that manipulates polarized light. ...

{main|Wollaston prism}
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 modification 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, the prism can be actively focused.

3. The two rays are focused by the condenser lens for passage through the sample. These two rays are focused so they will pass through two adjacent points in the sample, around 0.2μm apart.

The sample is effectively getting illuminated by two coherent 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.
The route of light througn a DIC microscope.
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The route of light througn a DIC microscope.

4. The rays travel through the different, adjacent, areas of the sample. They will experience different optical path lengths due to variation across the sample. 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. Look up Phase in Wiktionary, the free dictionary Phase may refer to: Phase (matter), a physically distinctive form of a substance, such as the solid, liquid, and gaseous states of ordinary matter Phase (waves), the time position (or angle in the complex plane) within a cycle of a periodic waveform...

The passage of many pairs of rays through pairs of adjacent points in the sample (and their absorbance, refraction 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 and are focused for the second Nomarski-modified Wollaston prism. The straw seems to be broken, due to refraction of light as it emerges into the air. ... Look up Phase in Wiktionary, the free dictionary Phase may refer to: Phase (matter), a physically distinctive form of a substance, such as the solid, liquid, and gaseous states of ordinary matter Phase (waves), the time position (or angle in the complex plane) within a cycle of a periodic waveform... An objective lens is the lens in a microscope, telescope, camera or other optical instrument, that receives the first light rays from the object being observed. ...


6. The second prism recombines the two rays into one polarised at 135°. The combination of the rays leads to interference, brightening or darkening the image at that point according to the optical path difference. Interference of two circular waves - Wavelength (decreasing bottom to top) and Wave centers distance (increasing to the right). ...

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 interfering with a ray of light that passed through the same point in the specimin, it will interfere with a ray of light that went through an adjacent point and 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 of seen.
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Differentiation can mean the following: In biology: cellular differentiation; evolutionary differentiation; In mathematics: see: derivative In cosmogony: planetary differentiation Differentiation (geology); Differentiation (logic); Differentiation (marketing). ...

The image

An illustration of the process of image production in a DIC microscope.
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An illustration of the process of image production in a DIC microscope.

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 defiend by the orientation of the Wollaston prisms.


As explained above the image is generated from two identical bright field images being overlayed slightly offset from each other (typically around 0.2μm), 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 eiter 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, if the phase difference at the joint between two substances is too large then the phase difference would reach 180° (half a wavelength), which is complete destructive interference, and then move on toward 360° (a full wavelength), where it would reach complete constructive interference, and an anomalous bright region.


It is worth noting 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, and so the differential of the refractive index (optical density) of the sample.

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Advantages and disadvantages

DIC has strong advantages in uses involving 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.


The main limitation of DIC is its requirement for a transparent sample of fairly similar refractive index to its surroundings. DIC is unsuitable (in biology) for thick samples, such as tissue slices, and highly pigmented cells. DIC is also unsuitable for most non biological uses because of its dependence on polarisation, which many physical samples would affect.

Orientation specific imaging in DIC.
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Orientation specific imaging in DIC.

Image quality, when used under suitable conditions, is outstanding in resolution and almost entirely free of artefacts. 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.

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See also

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It has been suggested that this article or section be merged into microscope. ... A phase contrast microscope is a microscope that does not require staining to view the slide. ...

References

  • Murphy, D., Differential interference contrast (DIC) microscopy and modulation contrast microscopy., 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
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External links


 
 

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