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Transmission electron microscopy (TEM) is a microscopy technique whereby a beam of electrons is transmitted through an ultra thin specimen, interacting with the specimen as it passes through it. An image is formed from the electrons transmitted through the specimen, magnified and focused by an objective lens and appears on an imaging screen, a fluorescent screen in most TEMs, plus a monitor, or on a layer of photographic film, or to be detected by a sensor such as a CCD camera. The first practical transmission electron microscope was built by Albert Prebus and James Hillier at the University of Toronto in 1938 using concepts developed earlier by Max Knoll and Ernst Ruska.
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History
Ernst Abbe originally proposed that the ability to resolve detail on an object was diffraction limit by the wavelength of the light used in imaging, thus limiting the useful obtainable magnification from an optical microscope to a few micron. Developments into UV microscopes, led by Koehler, allowed for an increase in resolving power of about a factor of two. However this required more expensive quartz optical components, due to the absorption of UV by glass. At this point it was believed that obtaining an image of sub-micron information was simply impossible due to this wavelength constraint.
It had earlier been recognised by Plücker in 1858 that the deflection of "cathode rays" (electrons) was possible by the use of magnetic fields[1]. This effect had been utilised to build primitive cathode ray oscilloscopes (CROs) as early as 1897 by Ferdinand Braun as a measurement device [2]. Indeed in 1891 it was recognised by Riecke that the cathode rays could be focussed by these magnetic fields, allowing for simple lens designs. Later this theory was extended by Hans Busch in his work published in 1926, who showed that the lens maker's equation, could under appropriate assumptions, be applicable to electrons[3]
In 1928, at the Technological University of Berlin Adolf Matthias, Professor of High voltage Technology and Electrical Installations, appointed Max Knoll to lead a team of researchers to advance the CRO. The team consisted of several PhD students including Ernst Ruska and Bodo von Borries. This team of researchers concerned themselves with lens design and CRO column placement, which they attempted to obtain the parameters that could be optimised to allow for construction of better CROs, as well as the development of electron optical components which could be used to generate low magnification (nearly 1:1) images. In 1931 the group successfully generated magnified images of mesh grids placed over the anode aperture. The device ussed two magnetic lenses to achieve higher magnifications, arguably the first electron microscope.
At this time the wave nature of electrons, which were considered charged matter particles, had not been fully realised until the publication of the De Broglie hypothesis in 1927. The group was unaware of this publication until 1932, where a chance comment by Houtermans brought the paper to their attention. It was quickly realised that the De Broglie wavelength of electrons was many orders of magnitude smaller than that for light, theoretically allowing for imaging at atomic scales. In April 1932, Ruska suggested the construction of a new electron microscope for direct imaging of specimens inserted into the microscope, rather than simple mesh grids or images of apertures. With this device successful diffraction and normal imaging of Aluminium sheet was achieved, however exceeding the magnification achievable with light microscopy had still not been successfully demonstrated. This goal was achieved in September 1933, using images of cotton fibres, which were quickly acquired before being damaged by the electron beam.
Research continued on the electron microscope at Siemens, which provided funding for the development of the electron microscope by von Borries and Ruska in 1936, the aim of the research to be the development of the microscope to improve it's imaging properties, particularly with regard to biological specimens. In 1939, the first commercial electron microscope, was installed in the Physics department of I.G Farben-Werke. A microscope of this series is now located at the Deutsches Museum in Munich. Further work on the electron microscope was hampered by the destruction of a a new laboratory constructed at Siemens by an air-raid, as well as the death of two of the researchers, Heinz Müller and Friedrick Krause during world war II.
Background
Theoretically the maximum resolution that one can obtain with a light microscope has been limited by the wavelength of the photons that are being used to probe the sample and the numerical aperture of the system. Early twentieth century scientists theorized ways of getting around the limitations of the relatively large wavelength of visible light (wavelengths of 400–700 nanometers) by using electrons. Like all matter, electrons have both wave and particle properties (as theorized by Louis-Victor de Broglie), and their wave-like properties mean that a beam of electrons can be made to behave like a beam of electromagnetic radiation. Electrons are usually generated in an electron microscope by a process known as thermionic emission from a filament, usually tungsten, in the same manner as a light bulb, or by field emission. The electrons are then accelerated by an electric potential (measured in V, or volts) and focused by electrostatic and electromagnetic lenses onto the sample. The transmitted beam contains information about electron density, phase and periodicity; this is used to form an image.
Components
A TEM is composed of several components, which include a vacuum system in which the electrons travel, an electron emission source for generation of the electron stream, a series of electromagnetic lenses, as well as electrostatic plates. The latter two allow the operator to guide and manipulate the beam as required.
Beam formation
From the top down, the TEM consists of an emission source, which may be a tungsten filament, or a lanthanum hexaboride (LaB6) source. This will be of the form of either a hairpin-style filament, or a small spike-shaped filament. By connecting this gun to an HV source (Typically ~120KV for many applications) the gun will, given sufficient current begin to emit electrons into the vacuum. This extraction is usually aided by the use of a Wehnelt cylinder. Once extracted, the upper lenses of the TEM allow for the formation of the electron probe to the desired size and location for later interaction with the sample.
Manipulation of the electron beam is performed using two physical effects. The interaction of electrons with a magnetic field will cause electrons to move according to the right hand rule, thus allowing for electromagnets to manipulate the electron beam. The use of magnetic fields allows for the formation of a magnetic lens of variable focussing power, the lens shape originating due to the distribution of magnetic flux. Additionally, electrostatic fields can cause the electrons to be deflected through a constant angle. Coupling of two deflections allows for the formation of a shift in the beam path, this being used in TEM for beam shifting, subsequently this is extremely important to STEM. From these two effects, as well as the use of an imaging system (such as a phosphor screen), sufficient control over the beam path is possible for TEM operation.
The lenses of a TEM allow for beam convergence, the angle of which can be varied, giving the TEM the ability to change magnification simply by modifying the amount of current that flows through the quadrupole or hexapole lenses. The quadrupole lens is an arrangement of electromagnetic coils at the vertices of the square, enabling the generation of a lensing magnetic fields, the hexapole configuration simply enhances the lens symmetry.
Typically a TEM consists of three stages of lensing, with many possible modifications on lens configurations, particularly in the application of energy filtered TEM. The stages are the condensor lenses, the objective lenses, and the projector lenses. The condensor lenses are responsible for primary beam formation, whilst the objective lenses focus the beam down onto the sample itself. The projector lenses are used to expand the beam onto the phosphor screen or other imaging device, such as film. The magnification of the TEM is due to the ratio of the distances between the specimen and the objective lens' image plane[4].
It is noted that TEM configurations differ significantly with implementation, with manufacturers using custom lens configurations, such as in spherical aberration corrected instruments, notably in high voltage field emission TEMs.
Imaging systems in a TEM consist of a phosphor screen for direct observation by the operator, and optionally an image recording system such as film based or phosphor screen coupled CCDs. Typically these devices can be removed or inserted into the beam path by the operator as required.
Imaging methods
Contrast Formation
Contrast formation in the TEM depends greatly on the mode of operation. Complex imaging techniques which utilise the unique ability to change lens strength or to deactivate a lens allows for many operating modes
Bright field
The most common mode of operation for a TEM utilises bright field imaging, whereby the contrast formation, when considered classically, can be considered to be formed directly by occlusion. Thicker regions of the sample, or regions with a higher atomic number will appear dark, whilst regions with no sample will appear bright -- hence the term "bright field".
Sample Staining
Details in a light microscope sample can be enhanced by stains that absorb light. Similarly TEM samples, usually of biological tissues, can utilise stains to enhance contrast. The stain absorbs electrons via phonon interaction, or scatters out of the part of the electron beam that is projected onto the imaging system. Compounds of heavy metals such as osmium, lead, or uranium may be used prior to TEM observation to selectively deposit electron dense atoms in or on the sample. During imaging beam electrons are deflected by electronic interactions with the positive atomic nuclei and negative electron clouds. Electron dense areas in the sample appear darker on the screen and on positive images.
Diffraction contrast
Samples can exhibit diffraction contrast, whereby the electron beam undergoes Bragg scattering, which in the case of a crystalline sample, disperses electrons into discrete locations in the back focal plane. By the placement of apertures in the back focal plane, i.e. the objective aperture, the desired Bragg reflections can be selected (or excluded), thus only parts of the sample that are causing the electrons to scatter to the selected reflections will end up projected onto the imaging apparatus. If the reflections that are selected do not include the unscattered beam (which will appear up at the focal point of the lens), then the image will appear dark wherever no sample scattering to the selected peak is present, as such a region without a specimen will appear dark. This is known as a dark-field image.
Modern TEMs are often equipped with specimen holders that allow the user to tilt the specimen to a range of angles in order to obtain specific diffraction conditions, and apertures placed above the specimen allow the user to select electrons that would otherwise be diffracted in a particular direction from entering the specimen.
By carefully selecting the orientation of the sample, it is possible not just to determine the position of defects but also to determine the type of defect present. If the sample is orientated so that one particular plane is only slightly tilted away from the strongest diffracting angle (known as the Bragg Angle), any distortion of the crystal plane that locally tilts the plane to the Bragg angle will produce particularly strong contrast variations. However, defects that produce only displacement of atoms that do not tilt the crystal to the Bragg angle (i.e. displacements parallel to the crystal plane) will not produce strong contrast.
Electron Energy Loss
Utilising the advanced technique of EELS, for TEMs appropriately equipped electrons can be rejected based upon their voltage (which, due to constant charge is their energy), using magnetic sector based devices known as EELS spectrometers. These devices allow for the selection of particular energy values, which can be associated with the way the electron has interacted with the sample. For example different elements in a sample result in different electron energies in the beam after the sample. This normally results in chromatic aberration -- however this effect can, for example, be used to generate an image which provides information on elemental composition.
Phase Contrast
Crystal structure can also be investigated by High Resolution Transmission Electron Microscopy (HRTEM), also known as phase contrast. When utilising a Field emission source, the images are formed due to differences in phase of electron waves, which is caused by specimen interaction. As image formation is given by the square of the incoming electron beam's complex modulus. As such, the image is not only dependent on the number of electrons hitting the screen, making direct interpretation of phase contrast images more complex. However this effect can be used to advantage, as it can be manipulated provide more information about the sample, such as in complex phase retrieval techniques.
Diffraction
As previously stated, by adjusting the magnetic lenses such that the back focal plane of the lens is placed on the imaging apparatus a diffraction pattern can be generated. For crystalline samples, this produces an image that consists of a series of dots in the case of a single crystal, or a series of rings in the case of a polycrystalline material. For the single crystal case the diffraction pattern is dependant upon the orientation of the specimen, and the resultant image is lattice image. This image provides the operator with information about the space group symmetries in the crystal and its orientation to the beam path, however this is typically done without utilising any information but the position at which the diffraction spots appear. Further analysis of this image is complex, ase the image is sensitive to a number of factors such as specimen thickness and orientation, objective lens defocus, spherical and chromatic aberration. Although quantitative interpretation of the contrast shown in lattice images is possible, it is inherently complicated and may require extensive computer simulation and analysis.
Modifications
The capabilities of the TEM can be further extended by additional stages and detectors, sometimes incorporated on the same microscope. An electron cryomicroscope is a TEM with a specimen holder capable of maintaining the specimen at liquid nitrogen or liquid helium temperatures. This allows imaging specimens prepared in vitreous ice, the preferred preparation technique for imaging individual molecules or macromolecular assemblies.
A TEM can be modified into a scanning transmission electron microscope (STEM) by the addition of a system that rasters the beam across the sample to form the image, combined with suitable detectors.
An analytical TEM is one equipped with detectors that can determine the elemental composition of the specimen by analysing its X-ray spectrum or the energy-loss spectrum of the transmitted electrons.
Modern research TEMs may include aberration correctors, to reduce the amount of distortion in the image, allowing information on features on the scale of 0.1 nm to be obtained (resolutions down to 0.05 nm have been achieved [5]) at magnifications of 50 million times [6]. Monochromators may also be used which reduce the energy spread of the incident electron beam to less than 0.15 eV. Major TEM makers include JEOL, Hitachi High-technologies, FEI Company (from merging with Philips Electron Optics) and Carl Zeiss.
Applications of the TEM
The TEM is used heavily in both material science/metallurgy and the biological sciences. In both cases the specimens must be very thin and able to withstand the high vacuum present inside the instrument.
For biological specimens, the maximum specimen thickness is roughly 1 micrometre. To withstand the instrument vacuum, biological specimens are typically held at liquid nitrogen temperatures after embedding in vitreous ice, or fixated using a negative staining material such as uranyl acetate or by plastic embedding. Typical biological applications include tomographic reconstructions of small cells or thin sections of larger cells and 3-D reconstructions of individual molecules via Single Particle Reconstruction.
In material science/metallurgy the specimens tend to be naturally resistant to vacuum, but must be prepared as a thin foil, or etched so some portion of the specimen is thin enough for the beam to penetrate. Preparation techniques to obtain an electron transparent region include ion beam milling and wedge polishing. The focused ion beam (FIB) is a relatively new technique to prepare thin samples for TEM examination from larger specimens. Because the FIB can be used to micro-machine samples very precisely, it is possible to mill very thin membranes from a specific area of a sample, such as a semiconductor or metal. Materials that have dimensions small enough to be electron transparent, such as powders or nanotubes, can be quickly produced by the deposition of a dilute sample containing the specimen onto support grids. The suspension is normally a volatile solvent, such as ethanol, ensuring that the solvent rapidly evaporates allowing a sample that can be rapidly analysed.
Limitations
There are a number of drawbacks to the TEM technique. Many materials require extensive sample preparation to produce a sample thin enough to be electron transparent, which makes TEM analysis a relatively time consuming process with a low throughput of samples. Graphene, a carbon nanomaterial, relatively transparent, very hard and just one atom thick, is currently being used as a platform on which the materials to be examined are placed. Being almost transparent to electrons, a graphene substrate has been able to show single hydrogen atom and hydrocarbons. The structure of the sample may also be changed during the preparation process. Also the field of view is relatively small, raising the possibility that the region analysed may not be characteristic of the whole sample. There is potential that the sample may be damaged by the electron beam, particularly in the case of biological materials.
Resolution limits
Resolution of the HRTEM is limited by spherical and chromatic aberration, but a new generation of aberration correctors has been able to overcome spherical aberration. Software correction of spherical aberration has allowed the production of images with sufficient resolution to show carbon atoms in diamond separated by only 0.89 ångströms and atoms in silicon at 0.78 ångströms (78 pm) at magnifications of 50 million times. Improved resolution has also allowed the imaging of lighter atoms that scatter electrons less efficiently — lithium atoms have been imaged in lithium battery materials[7]. The ability to determine the positions of atoms within materials has made the HRTEM an indispensable tool for nanotechnology research and development in many fields, including heterogeneous catalysis and the development of semiconductor devices for electronics and photonics.
See also
- Scanning electron microscope
- Electron microscope
- Transmission Electron Aberration-corrected Microscope
- Energy filtered transmission electron microscopy
- Electron diffraction
References
- ^ Plücker, J. (1858). "„Über die Einwirkung des Magneten auf die elektrischen Entladungen in verdünnten Gasen “". Poggendorffs Annalen der Physik und Chemie 103: 88--106.
- ^ "Ferdinand Braun, The Nobel Prize in Physics 1909, Biography".
- ^ ""The Nobel Prize in Physics 1986, Perspectives Perspectives - Life through a Lens"".
- ^ "The objective lens of a TEM, the heart of the electron microscope".
- ^ TEAM Project Achieves Microscopy Breakthrough
- ^ The Scale of Things (Office of Basic Energy Sciences)
- ^ "http://www.fei.com/Portals/_default/PDFs/content/2006_06_LithiumImagingOkeefe_wp.pdf Imaging lithium atoms at sub-Ångström resolution]".
External links
- The National Center for Electron Microscopy, Berkeley California USA
- The National Center for Macromolecular Imaging, Houston Texas USA
- The National Resource for Automated Molecular Microscopy, La Jolla California USA
- Manufacturer of TEM's (FEI Company)
- Delong Group
- Tutorial courses in Transmission Electron Microscopy
- Cambridge University Teaching and Learning Package on TEM
Wikipedia content modification information:
- This page was last modified on 6 September 2008, at 04:29.
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