Phase-contrast imaging
Encyclopedia
Phase-contrast imaging is a method of imaging
that has a range of different applications. It exploits differences in the refractive index of different materials to differentiate between structures under analysis. In conventional light microscopy, phase contrast can be employed to distinguish between structures of similar transparency, and to examine crystals on the basis of their double refraction
. This has uses in biological, medical and geological science. In X-ray tomography, the same physical principles can be used to increase image contrast by highlighting small details of differing refractive index within structures that are otherwise uniform. In transmission electron microscopy
(TEM), phase contrast enables very high resolution (HR) imaging (at resolutions below one angstrom
), making it possible to distinguish individual atoms from each other by their different refractive indices.
, and so bend light and delay its passage through the sample by different amounts. The retardation of the light results in some waves being 'out of phase' with others, and so to the human eye a microscope in phase contrast mode effectively darkens or brightens particular areas to reflect this change.
Phase contrast is used extensively in optical microscopy, in both biological and geological sciences. In biology, it is employed in viewing unstained biological samples
with the human eye, making it possible to distinguish between structures that are of very similar transparency.
In geology, phase contrast is exploited in a different way to highlight differences between mineral crystals cut to a standardised thin section (usually 30 microns) and mounted under a light microscope. Crystalline materials are capable of exhibiting double refraction
, in which light rays entering a crystal are split into two beams that may exhibit different refractive indices, depending on the angle
at which they enter the crystal. The phase contrast between the two rays can be detected with the human eye using particular optical filters. As the exact nature of the double refraction varies for different crystal structures, phase contrast aids in the identification of minerals.
Propagation-based phase contrast uses free-space propagation
to get edge enhancement, talbot
interferometry uses a set of diffraction grating
s to measure the derivative of the phase, refraction-enhanced imaging uses an analyzer crystal also for differential measurement, and x-ray interferometry
uses a crystal interferometer to measure the phase directly. The advantage of these methods compared to normal absorption-contrast x-ray imaging is the higher contrast making it possible to see smaller details. One disadvantage is that these methods require more sophisticated equipment, such as synchrotron
or microfocus x-ray sources, x-ray optics
and high resolution x-ray detectors. The high demands on equipment are mainly due to the small variations in refractive index for x-rays. The refractive index is normally smaller than 1 with a difference from 1 between and .
All of these methods produce images that can be used to calculate the projection
s (integrals) of the refractive index in the imaging direction. For propagation-based phase contrast there are phase-retrieval
algorithms, for talbot interferometry and refraction-enhanced imaging the image is integrated in the proper direction, and for x-ray interferometry phase unwrapping is performed. For this reason they are well suited for tomography
, i.e. reconstruction of a 3D-map of the refractive index of the object from many images at slightly different angles. For x-ray radiation the difference from 1 of the refractive index is essentially proportional to the density
of the material.
Synchrotron X-ray tomography can employ phase contrast imaging to enable imaging of the interior surfaces of objects. In this context, phase contrast imaging is used to enhance the contrast that would normally be possible from conventional radiographic imaging. A difference in the refractive index between a detail and its surroundings causes a phase shift between the light wave that travels through the detail and that which travels outside the detail. An interference pattern results, marking out the detail.
This method has been used to image Precambrian
metazoan embryo
s from the Doushantuo Formation in China, allowing the internal structure of delicate microfossils to be imaged without destroying the original specimen.
, phase contrast imaging may be employed to image columns of individual atoms. This ability arises from the fact that the atoms in a material diffract electrons as the electrons pass through them (the relative phases of the electrons change upon transmission through the sample), causing diffraction
contrast in addition to the already present contrast in the transmitted beam. Phase-contrast imaging is the highest resolution
imaging technique ever developed, and can allow for resolutions of less than one angstrom
(less than 0.1 nanometres). It thus enables the direct viewing of columns of atoms in a crystalline material.
The interpretation of phase-contrast images is not a straightforward task. Deconvolving the contrast seen in an HR image to determine which features are due to which atoms in the material can rarely, if ever, be done by eye. Instead, because the combination of contrasts due to multiple diffracting elements and planes and the transmitted beam
is complex, computer simulations are used to determine what sort of contrast different structures may produce in a phase-contrast image. Thus, a reasonable amount of information about the sample needs to be understood before a phase contrast image can be properly interpreted, such as a conjecture as to what crystal structure
the material has.
Phase-contrast images are formed by removing the objective
aperture
entirely or by using a very large objective aperture. This ensures that not only the transmitted beam, but also the diffracted ones are allowed to contribute to the image. Instruments that are specifically designed for phase-contrast imaging are often called HRTEMs (high resolution transmission electron microscopes), and differ from analytical TEMs mainly in the design of the electron beam column. Whereas analytical TEMs employ additional detectors attached to the column for spectroscopic measurements
, HRTEMs have little or no additional attachments so as to ensure a uniform electromagnetic
environment all the way down the column for each beam leaving the sample (transmitted and diffracted). Because phase-contrast imaging relies on differences in phase between electrons leaving the sample, any additional phase shifts that occur between the sample and the viewing screen can make the image impossible to interpret. Thus, a very low degree of lens aberration
is also a requirement for HRTEMs, and advances in spherical aberration
(Cs) correction have enabled a new generation of HRTEMs to reach resolutions once thought impossible.
See also=
Image
An image is an artifact, for example a two-dimensional picture, that has a similar appearance to some subject—usually a physical object or a person.-Characteristics:...
that has a range of different applications. It exploits differences in the refractive index of different materials to differentiate between structures under analysis. In conventional light microscopy, phase contrast can be employed to distinguish between structures of similar transparency, and to examine crystals on the basis of their double refraction
Birefringence
Birefringence, or double refraction, is the decomposition of a ray of light into two rays when it passes through certain anisotropic materials, such as crystals of calcite or boron nitride. The effect was first described by the Danish scientist Rasmus Bartholin in 1669, who saw it in calcite...
. This has uses in biological, medical and geological science. In X-ray tomography, the same physical principles can be used to increase image contrast by highlighting small details of differing refractive index within structures that are otherwise uniform. In transmission electron microscopy
Transmission electron microscopy
Transmission electron microscopy is a microscopy technique whereby a beam of electrons is transmitted through an ultra thin specimen, interacting with the specimen as it passes through...
(TEM), phase contrast enables very high resolution (HR) imaging (at resolutions below one angstrom
Ångström
The angstrom or ångström, is a unit of length equal to 1/10,000,000,000 of a meter . Its symbol is the Swedish letter Å....
), making it possible to distinguish individual atoms from each other by their different refractive indices.
Light microscopy
Phase contrast takes advantage of the fact that different structures have different refractive indicesRefractive index
In optics the refractive index or index of refraction of a substance or medium is a measure of the speed of light in that medium. It is expressed as a ratio of the speed of light in vacuum relative to that in the considered medium....
, and so bend light and delay its passage through the sample by different amounts. The retardation of the light results in some waves being 'out of phase' with others, and so to the human eye a microscope in phase contrast mode effectively darkens or brightens particular areas to reflect this change.
Phase contrast is used extensively in optical microscopy, in both biological and geological sciences. In biology, it is employed in viewing unstained biological samples
Phase contrast microscopy
Phase contrast microscopy is an optical microscopy illumination technique of great importance to biologists in which small phase shifts in the light passing through a transparent specimen are converted into amplitude or contrast changes in the image.A phase contrast microscope does not require...
with the human eye, making it possible to distinguish between structures that are of very similar transparency.
In geology, phase contrast is exploited in a different way to highlight differences between mineral crystals cut to a standardised thin section (usually 30 microns) and mounted under a light microscope. Crystalline materials are capable of exhibiting double refraction
Birefringence
Birefringence, or double refraction, is the decomposition of a ray of light into two rays when it passes through certain anisotropic materials, such as crystals of calcite or boron nitride. The effect was first described by the Danish scientist Rasmus Bartholin in 1669, who saw it in calcite...
, in which light rays entering a crystal are split into two beams that may exhibit different refractive indices, depending on the angle
Optic axis of a crystal
The optic axis of a crystal is the direction in which a ray of transmitted light suffers no birefringence . Due to the internal structure of the crystal , light propagates along the optical axis differently than in other directions...
at which they enter the crystal. The phase contrast between the two rays can be detected with the human eye using particular optical filters. As the exact nature of the double refraction varies for different crystal structures, phase contrast aids in the identification of minerals.
X-ray imaging
There are four main techniques for x-ray phase-contrast imaging, all utilizing different principles to convert phase variations in the x-rays emerging from the object, into intensity variations at an x-ray detector.Propagation-based phase contrast uses free-space propagation
Wave propagation
Wave propagation is any of the ways in which waves travel.With respect to the direction of the oscillation relative to the propagation direction, we can distinguish between longitudinal wave and transverse waves....
to get edge enhancement, talbot
Talbot effect
The Talbot effect is a near-field diffraction effect first observed in 1836 by Henry Fox Talbot. When a laterally periodic wave distribution is incident upon a diffraction grating,...
interferometry uses a set of diffraction grating
Diffraction grating
In optics, a diffraction grating is an optical component with a periodic structure, which splits and diffracts light into several beams travelling in different directions. The directions of these beams depend on the spacing of the grating and the wavelength of the light so that the grating acts as...
s to measure the derivative of the phase, refraction-enhanced imaging uses an analyzer crystal also for differential measurement, and x-ray interferometry
Interferometry
Interferometry refers to a family of techniques in which electromagnetic waves are superimposed in order to extract information about the waves. An instrument used to interfere waves is called an interferometer. Interferometry is an important investigative technique in the fields of astronomy,...
uses a crystal interferometer to measure the phase directly. The advantage of these methods compared to normal absorption-contrast x-ray imaging is the higher contrast making it possible to see smaller details. One disadvantage is that these methods require more sophisticated equipment, such as synchrotron
Synchrotron
A synchrotron is a particular type of cyclic particle accelerator in which the magnetic field and the electric field are carefully synchronised with the travelling particle beam. The proton synchrotron was originally conceived by Sir Marcus Oliphant...
or microfocus x-ray sources, x-ray optics
X-ray optics
X-ray optics is the branch of optics which manipulates X-rays instead of visible light. While lenses for visible light are made of transparent materials that can have a refractive index substantially larger than 1, for X-rays the index of refraction is slightly smaller than unity. The principal...
and high resolution x-ray detectors. The high demands on equipment are mainly due to the small variations in refractive index for x-rays. The refractive index is normally smaller than 1 with a difference from 1 between and .
All of these methods produce images that can be used to calculate the projection
Parallel projection
Parallel projections have lines of projection that are parallel both in reality and in the projection plane.Parallel projection corresponds to a perspective projection with an infinite focal length , or "zoom".Within parallel projection there is an ancillary category known as "pictorials"...
s (integrals) of the refractive index in the imaging direction. For propagation-based phase contrast there are phase-retrieval
Phase retrieval
Phase retrieval concerns the solution to the phase problem. Given a complex signal F, of amplitude |F|, and phase \phi:phase retrieval consists in finding the phase that for a measured amplitude satisfies a set of constraints....
algorithms, for talbot interferometry and refraction-enhanced imaging the image is integrated in the proper direction, and for x-ray interferometry phase unwrapping is performed. For this reason they are well suited for tomography
Tomography
Tomography refers to imaging by sections or sectioning, through the use of any kind of penetrating wave. A device used in tomography is called a tomograph, while the image produced is a tomogram. The method is used in radiology, archaeology, biology, geophysics, oceanography, materials science,...
, i.e. reconstruction of a 3D-map of the refractive index of the object from many images at slightly different angles. For x-ray radiation the difference from 1 of the refractive index is essentially proportional to the density
Density
The mass density or density of a material is defined as its mass per unit volume. The symbol most often used for density is ρ . In some cases , density is also defined as its weight per unit volume; although, this quantity is more properly called specific weight...
of the material.
Synchrotron X-ray tomography can employ phase contrast imaging to enable imaging of the interior surfaces of objects. In this context, phase contrast imaging is used to enhance the contrast that would normally be possible from conventional radiographic imaging. A difference in the refractive index between a detail and its surroundings causes a phase shift between the light wave that travels through the detail and that which travels outside the detail. An interference pattern results, marking out the detail.
This method has been used to image Precambrian
Precambrian
The Precambrian is the name which describes the large span of time in Earth's history before the current Phanerozoic Eon, and is a Supereon divided into several eons of the geologic time scale...
metazoan embryo
Embryo
An embryo is a multicellular diploid eukaryote in its earliest stage of development, from the time of first cell division until birth, hatching, or germination...
s from the Doushantuo Formation in China, allowing the internal structure of delicate microfossils to be imaged without destroying the original specimen.
Transmission electron microscopy
In the field of Transmission Electron MicroscopyTransmission electron microscopy
Transmission electron microscopy is a microscopy technique whereby a beam of electrons is transmitted through an ultra thin specimen, interacting with the specimen as it passes through...
, phase contrast imaging may be employed to image columns of individual atoms. This ability arises from the fact that the atoms in a material diffract electrons as the electrons pass through them (the relative phases of the electrons change upon transmission through the sample), causing diffraction
Diffraction
Diffraction refers to various phenomena which occur when a wave encounters an obstacle. Italian scientist Francesco Maria Grimaldi coined the word "diffraction" and was the first to record accurate observations of the phenomenon in 1665...
contrast in addition to the already present contrast in the transmitted beam. Phase-contrast imaging is the highest resolution
Image resolution
Image resolution is an umbrella term that describes the detail an image holds. The term applies to raster digital images, film images, and other types of images. Higher resolution means more image detail....
imaging technique ever developed, and can allow for resolutions of less than one angstrom
Ångström
The angstrom or ångström, is a unit of length equal to 1/10,000,000,000 of a meter . Its symbol is the Swedish letter Å....
(less than 0.1 nanometres). It thus enables the direct viewing of columns of atoms in a crystalline material.
The interpretation of phase-contrast images is not a straightforward task. Deconvolving the contrast seen in an HR image to determine which features are due to which atoms in the material can rarely, if ever, be done by eye. Instead, because the combination of contrasts due to multiple diffracting elements and planes and the transmitted beam
Light beam
A light beam or beam of light is a narrow projection of light energy radiating from a source into a beam. Sunlight is a natural example of a light beam when filtered through various mediums...
is complex, computer simulations are used to determine what sort of contrast different structures may produce in a phase-contrast image. Thus, a reasonable amount of information about the sample needs to be understood before a phase contrast image can be properly interpreted, such as a conjecture as to what crystal structure
Crystal structure
In mineralogy and crystallography, crystal structure is a unique arrangement of atoms or molecules in a crystalline liquid or solid. A crystal structure is composed of a pattern, a set of atoms arranged in a particular way, and a lattice exhibiting long-range order and symmetry...
the material has.
Phase-contrast images are formed by removing the objective
Objective (optics)
In an optical instrument, the objective is the optical element that gathers light from the object being observed and focuses the light rays to produce a real image. Objectives can be single lenses or mirrors, or combinations of several optical elements. They are used in microscopes, telescopes,...
aperture
Aperture
In optics, an aperture is a hole or an opening through which light travels. More specifically, the aperture of an optical system is the opening that determines the cone angle of a bundle of rays that come to a focus in the image plane. The aperture determines how collimated the admitted rays are,...
entirely or by using a very large objective aperture. This ensures that not only the transmitted beam, but also the diffracted ones are allowed to contribute to the image. Instruments that are specifically designed for phase-contrast imaging are often called HRTEMs (high resolution transmission electron microscopes), and differ from analytical TEMs mainly in the design of the electron beam column. Whereas analytical TEMs employ additional detectors attached to the column for spectroscopic measurements
Spectroscopy
Spectroscopy is the study of the interaction between matter and radiated energy. Historically, spectroscopy originated through the study of visible light dispersed according to its wavelength, e.g., by a prism. Later the concept was expanded greatly to comprise any interaction with radiative...
, HRTEMs have little or no additional attachments so as to ensure a uniform electromagnetic
Electromagnetic radiation
Electromagnetic radiation is a form of energy that exhibits wave-like behavior as it travels through space...
environment all the way down the column for each beam leaving the sample (transmitted and diffracted). Because phase-contrast imaging relies on differences in phase between electrons leaving the sample, any additional phase shifts that occur between the sample and the viewing screen can make the image impossible to interpret. Thus, a very low degree of lens aberration
Aberration in optical systems
Aberrations are departures of the performance of an optical system from the predictions of paraxial optics. Aberration leads to blurring of the image produced by an image-forming optical system. It occurs when light from one point of an object after transmission through the system does not converge...
is also a requirement for HRTEMs, and advances in spherical aberration
Spherical aberration
thumb|right|Spherical aberration. A perfect lens focuses all incoming rays to a point on the [[Optical axis|optic axis]]. A real lens with spherical surfaces suffers from spherical aberration: it focuses rays more tightly if they enter it far from the optic axis than if they enter closer to the...
(Cs) correction have enabled a new generation of HRTEMs to reach resolutions once thought impossible.
See also=
- Refractive indexRefractive indexIn optics the refractive index or index of refraction of a substance or medium is a measure of the speed of light in that medium. It is expressed as a ratio of the speed of light in vacuum relative to that in the considered medium....
- MicroscopyMicroscopyMicroscopy is the technical field of using microscopes to view samples and objects that cannot be seen with the unaided eye...
- Phase contrast microscopyPhase contrast microscopyPhase contrast microscopy is an optical microscopy illumination technique of great importance to biologists in which small phase shifts in the light passing through a transparent specimen are converted into amplitude or contrast changes in the image.A phase contrast microscope does not require...
- X-ray computed tomography
- High Resolution Transmission Electron MicroscopyHigh Resolution Transmission Electron MicroscopyHigh-resolution transmission electron microscopy is an imaging mode of the transmission electron microscope that allows the imaging of the crystallographic structure of a sample at an atomic scale. Because of its high resolution, it is an invaluable tool to study nanoscale properties of...