Electron tomography

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Basic principle of tomography: superposition free tomographic cross sections S1 and S2 compared with the projected image P TomographyPrinciple Illustration.png
Basic principle of tomography: superposition free tomographic cross sections S1 and S2 compared with the projected image P

Electron tomography (ET) is a tomography technique for obtaining detailed 3D structures [1] of sub-cellular, macro-molecular, or materials specimens. Electron tomography is an extension of traditional transmission electron microscopy and uses a transmission electron microscope to collect the data. In the process, a beam of electrons is passed through the sample at incremental degrees of rotation around the center of the target sample. This information is collected and used to assemble a three-dimensional image of the target. For biological applications, the typical resolution of ET systems [2] are in the 5–20 nm range, suitable for examining supra-molecular multi-protein structures, although not the secondary and tertiary structure of an individual protein or polypeptide. [3] [4] Recently, atomic resolution in 3D electron tomography reconstructions has been demonstrated. [5] [6]

Contents

BF-TEM and ADF-STEM tomography

In the field of biology, bright-field transmission electron microscopy (BF-TEM) and high-resolution TEM (HRTEM) are the primary imaging methods for tomography tilt series acquisition. However, there are two issues associated with BF-TEM and HRTEM. First, acquiring an interpretable 3-D tomogram requires that the projected image intensities vary monotonically with material thickness. This condition is difficult to guarantee in BF/HRTEM, where image intensities are dominated by phase-contrast with the potential for multiple contrast reversals with thickness, making it difficult to distinguish voids from high-density inclusions. [7] [8] Second, the contrast transfer function of BF-TEM is essentially a high-pass filter – information at low spatial frequencies is significantly suppressed – resulting in an exaggeration of sharp features. However, the technique of annular dark-field scanning transmission electron microscopy (ADF-STEM), which is typically used on material specimens, [9] more effectively suppresses phase and diffraction contrast, providing image intensities that vary with the projected mass-thickness of samples up to micrometres thick for materials with low atomic number. ADF-STEM also acts as a low-pass filter, eliminating the edge-enhancing artifacts common in BF/HRTEM. Thus, provided that the features can be resolved, ADF-STEM tomography can yield a reliable reconstruction of the underlying specimen which is extremely important for its application in materials science. [10] For 3D imaging, the resolution is traditionally described by the Crowther criterion. In 2010, a 3D resolution of 0.5±0.1×0.5±0.1×0.7±0.2 nm was achieved with a single-axis ADF-STEM tomography. [11]

Atomic Electron Tomography (AET)

Schematic showing the concept of electron tomography. Electron Tomography.tif
Schematic showing the concept of electron tomography.

Atomic level resolution in 3D electron tomography reconstructions has been demonstrated. Reconstructions of crystal defects such as stacking faults, grain boundaries, dislocations, and twinning in structures have been achieved. [12] This method is relevant to the physical sciences, where cryo-EM techniques cannot always be used to locate the coordinates of individual atoms in disordered materials. AET reconstructions are achieved using the combination of an ADF-STEM tomographic tilt series and iterative algorithms for reconstruction. Currently, algorithms such as the real-space algebraic reconstruction technique (ART) and the fast Fourier transform equal slope tomography (EST) are used to address issues such as image noise, sample drift, and limited data. [13] ADF-STEM tomography has recently been used to directly visualize the atomic structure of screw dislocations in nanoparticles. [14] [15] [16] [17] AET has also been used to find the 3D coordinates of 3,769 atoms in a tungsten needle with 19 pm precision [18] and 20,000 atoms in a multiply twinned palladium nanoparticle. [19] The combination of AET with electron energy loss spectroscopy (EELS) allows for investigation of electronic states in addition to 3D reconstruction. [20] [21] Challenges to atomic level resolution from electron tomography include the need for better reconstruction algorithms and increased precision of tilt angle required to image defects in non-crystalline samples.

Different tilting methods

The most popular tilting methods are the single-axis and the dual-axis tilting methods. The geometry of most specimen holders and electron microscopes normally precludes tilting the specimen through a full 180° range, which can lead to artifacts in the 3D reconstruction of the target. [22] [23] Standard single-tilt sample holders have a limited rotation of ±80°, leading to a missing wedge in the reconstruction. A solution is to use needle shaped-samples to allow for full rotation. By using dual-axis tilting, the reconstruction artifacts are reduced by a factor of compared to single-axis tilting. However, twice as many images need to be taken. Another method of obtaining a tilt-series is the so-called conical tomography method, in which the sample is tilted, and then rotated a complete turn. [24]

See also

Related Research Articles

<span class="mw-page-title-main">Electron microscope</span> Type of microscope with electrons as a source of illumination

An electron microscope is a microscope that uses a beam of electrons as a source of illumination. They use electron optics that are analogous to the glass lenses of an optical light microscope to control the electron beam, for instance focusing them to produce magnified images or electron diffraction patterns. As the wavelength of an electron can be up to 100,000 times smaller than that of visible light, electron microscopes have a much higher resolution of about 0.1 nm, which compares to about 200 nm for light microscopes. Electron microscope may refer to:

<span class="mw-page-title-main">Transmission electron microscopy</span> Imaging and diffraction using electrons that pass through samples

Transmission electron microscopy (TEM) is a microscopy technique in which a beam of electrons is transmitted through a specimen to form an image. The specimen is most often an ultrathin section less than 100 nm thick or a suspension on a grid. An image is formed from the interaction of the electrons with the sample as the beam is transmitted through the specimen. The image is then magnified and focused onto an imaging device, such as a fluorescent screen, a layer of photographic film, or a detector such as a scintillator attached to a charge-coupled device or a direct electron detector.

<span class="mw-page-title-main">Tomography</span> Imaging by sections or sectioning using a penetrative wave

Tomography is imaging by sections or sectioning that uses any kind of penetrating wave. The method is used in radiology, archaeology, biology, atmospheric science, geophysics, oceanography, plasma physics, materials science, cosmochemistry, astrophysics, quantum information, and other areas of science. The word tomography is derived from Ancient Greek τόμος tomos, "slice, section" and γράφω graphō, "to write" or, in this context as well, "to describe." A device used in tomography is called a tomograph, while the image produced is a tomogram.

<span class="mw-page-title-main">Electron backscatter diffraction</span> Scanning electron microscopy technique

Electron backscatter diffraction (EBSD) is a scanning electron microscopy (SEM) technique used to study the crystallographic structure of materials. EBSD is carried out in a scanning electron microscope equipped with an EBSD detector comprising at least a phosphorescent screen, a compact lens and a low-light camera. In the microscope an incident beam of electrons hits a tilted sample. As backscattered electrons leave the sample, they interact with the atoms and are both elastically diffracted and lose energy, leaving the sample at various scattering angles before reaching the phosphor screen forming Kikuchi patterns (EBSPs). The EBSD spatial resolution depends on many factors, including the nature of the material under study and the sample preparation. They can be indexed to provide information about the material's grain structure, grain orientation, and phase at the micro-scale. EBSD is used for impurities and defect studies, plastic deformation, and statistical analysis for average misorientation, grain size, and crystallographic texture. EBSD can also be combined with energy-dispersive X-ray spectroscopy (EDS), cathodoluminescence (CL), and wavelength-dispersive X-ray spectroscopy (WDS) for advanced phase identification and materials discovery.

Electron crystallography is a method to determine the arrangement of atoms in solids using a transmission electron microscope (TEM). It can involve the use of high-resolution transmission electron microscopy images, electron diffraction patterns including convergent-beam electron diffraction or combinations of these. It has been successful in determining some bulk structures, and also surface structures. Two related methods are low-energy electron diffraction which has solved the structure of many surfaces, and reflection high-energy electron diffraction which is used to monitor surfaces often during growth.

<span class="mw-page-title-main">Scanning transmission electron microscopy</span> Scanning microscopy using thin samples and transmitted electrons

A scanning transmission electron microscope (STEM) is a type of transmission electron microscope (TEM). Pronunciation is [stɛm] or [ɛsti:i:ɛm]. As with a conventional transmission electron microscope (CTEM), images are formed by electrons passing through a sufficiently thin specimen. However, unlike CTEM, in STEM the electron beam is focused to a fine spot which is then scanned over the sample in a raster illumination system constructed so that the sample is illuminated at each point with the beam parallel to the optical axis. The rastering of the beam across the sample makes STEM suitable for analytical techniques such as Z-contrast annular dark-field imaging, and spectroscopic mapping by energy dispersive X-ray (EDX) spectroscopy, or electron energy loss spectroscopy (EELS). These signals can be obtained simultaneously, allowing direct correlation of images and spectroscopic data.

<span class="mw-page-title-main">X-ray microtomography</span> X-ray 3D imaging method

In radiography, X-ray microtomography uses X-rays to create cross-sections of a physical object that can be used to recreate a virtual model without destroying the original object. It is similar to tomography and X-ray computed tomography. The prefix micro- is used to indicate that the pixel sizes of the cross-sections are in the micrometre range. These pixel sizes have also resulted in creation of its synonyms high-resolution X-ray tomography, micro-computed tomography, and similar terms. Sometimes the terms high-resolution computed tomography (HRCT) and micro-CT are differentiated, but in other cases the term high-resolution micro-CT is used. Virtually all tomography today is computed tomography.

<span class="mw-page-title-main">Transmission electron cryomicroscopy</span>

Transmission electron cryomicroscopy (CryoTEM), commonly known as cryo-EM, is a form of cryogenic electron microscopy, more specifically a type of transmission electron microscopy (TEM) where the sample is studied at cryogenic temperatures. Cryo-EM, specifically 3-dimensional electron microscopy (3DEM), is gaining popularity in structural biology.

Phase-contrast imaging is a method of imaging that has a range of different applications. It measures 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, making it possible to distinguish features a few Angstrom apart.

<span class="mw-page-title-main">Focused ion beam</span> Device

Focused ion beam, also known as FIB, is a technique used particularly in the semiconductor industry, materials science and increasingly in the biological field for site-specific analysis, deposition, and ablation of materials. A FIB setup is a scientific instrument that resembles a scanning electron microscope (SEM). However, while the SEM uses a focused beam of electrons to image the sample in the chamber, a FIB setup uses a focused beam of ions instead. FIB can also be incorporated in a system with both electron and ion beam columns, allowing the same feature to be investigated using either of the beams. FIB should not be confused with using a beam of focused ions for direct write lithography. These are generally quite different systems where the material is modified by other mechanisms.

<span class="mw-page-title-main">Cryogenic electron tomography</span>

Cryogenic electron tomography (cryoET) is an imaging technique used to reconstruct high-resolution (~1–4 nm) three-dimensional volumes of samples, often biological macromolecules and cells. cryoET is a specialized application of transmission electron cryomicroscopy (CryoTEM) in which samples are imaged as they are tilted, resulting in a series of 2D images that can be combined to produce a 3D reconstruction, similar to a CT scan of the human body. In contrast to other electron tomography techniques, samples are imaged under cryogenic conditions. For cellular material, the structure is immobilized in non-crystalline, vitreous ice, allowing them to be imaged without dehydration or chemical fixation, which would otherwise disrupt or distort biological structures.

<span class="mw-page-title-main">Optical sectioning</span> Imaging of focal planes within a thick sample

Optical sectioning is the process by which a suitably designed microscope can produce clear images of focal planes deep within a thick sample. This is used to reduce the need for thin sectioning using instruments such as the microtome. Many different techniques for optical sectioning are used and several microscopy techniques are specifically designed to improve the quality of optical sectioning.

A Low-voltage electron microscope (LVEM) is an electron microscope which operates at accelerating voltages of a few kiloelectronvolts (keV) or less. Traditional electron microscopes use accelerating voltages in the range of 10-1000 keV.

<span class="mw-page-title-main">Single particle analysis</span> Method of analyzing transmission electron microscopy imagery

Single particle analysis is a group of related computerized image processing techniques used to analyze images from transmission electron microscopy (TEM). These methods were developed to improve and extend the information obtainable from TEM images of particulate samples, typically proteins or other large biological entities such as viruses. Individual images of stained or unstained particles are very noisy, and so hard to interpret. Combining several digitized images of similar particles together gives an image with stronger and more easily interpretable features. An extension of this technique uses single particle methods to build up a three-dimensional reconstruction of the particle. Using cryo-electron microscopy it has become possible to generate reconstructions with sub-nanometer resolution and near-atomic resolution first in the case of highly symmetric viruses, and now in smaller, asymmetric proteins as well. Single particle analysis can also be performed by inductively coupled plasma mass spectrometry (ICP-MS).

<span class="mw-page-title-main">Crystallographic image processing</span>

Crystallographic image processing (CIP) is traditionally understood as being a set of key steps in the determination of the atomic structure of crystalline matter from high-resolution electron microscopy (HREM) images obtained in a transmission electron microscope (TEM) that is run in the parallel illumination mode. The term was created in the research group of Sven Hovmöller at Stockholm University during the early 1980s and became rapidly a label for the "3D crystal structure from 2D transmission/projection images" approach. Since the late 1990s, analogous and complementary image processing techniques that are directed towards the achieving of goals with are either complementary or entirely beyond the scope of the original inception of CIP have been developed independently by members of the computational symmetry/geometry, scanning transmission electron microscopy, scanning probe microscopy communities, and applied crystallography communities.

<span class="mw-page-title-main">Cryogenic electron microscopy</span> Form of transmission electron microscopy (TEM)

Cryogenic electron microscopy (cryo-EM) is a cryomicroscopy technique applied on samples cooled to cryogenic temperatures. For biological specimens, the structure is preserved by embedding in an environment of vitreous ice. An aqueous sample solution is applied to a grid-mesh and plunge-frozen in liquid ethane or a mixture of liquid ethane and propane. While development of the technique began in the 1970s, recent advances in detector technology and software algorithms have allowed for the determination of biomolecular structures at near-atomic resolution. This has attracted wide attention to the approach as an alternative to X-ray crystallography or NMR spectroscopy for macromolecular structure determination without the need for crystallization.

4D scanning transmission electron microscopy is a subset of scanning transmission electron microscopy (STEM) which utilizes a pixelated electron detector to capture a convergent beam electron diffraction (CBED) pattern at each scan location. This technique captures a 2 dimensional reciprocal space image associated with each scan point as the beam rasters across a 2 dimensional region in real space, hence the name 4D STEM. Its development was enabled by evolution in STEM detectors and improvements computational power. The technique has applications in visual diffraction imaging, phase orientation and strain mapping, phase contrast analysis, among others.

<span class="mw-page-title-main">Transmission Kikuchi diffraction</span> Nanoscale orientation mapping method

Transmission Kikuchi Diffraction (TKD), also sometimes called transmission-electron backscatter diffraction (t-EBSD), is a method for orientation mapping at the nanoscale. It’s used for analysing the microstructures of thin transmission electron microscopy (TEM) specimens in the scanning electron microscope (SEM). This technique has been widely utilised in the characterization of nano-crystalline materials, including oxides, superconductors, and metallic alloys.

Dark-field X-ray microscopy is an imaging technique used for multiscale structural characterisation. It is capable of mapping deeply embedded structural elements with nm-resolution using synchrotron X-ray diffraction-based imaging. The technique works by using scattered X-rays to create a high degree of contrast, and by measuring the intensity and spatial distribution of the diffracted beams, it is possible to obtain a three-dimensional map of the sample's structure, orientation, and local strain.

<span class="mw-page-title-main">Jianwei Miao</span> Chinese-American physicist

Jianwei (John) Miao is a Professor in the Department of Physics and Astronomy and the California NanoSystems Institute at the University of California, Los Angeles. He performed the first experiment on extending crystallography to allow structural determination of non-crystalline specimens in 1999, which has been known as coherent diffractive imaging (CDI), lensless imaging, or computational microscopy. In 2012, Miao applied the CDI method to pioneer atomic electron tomography (AET), enabling the first determination of 3D atomic structures without assuming crystallinity or averaging.

References

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