Electron microscope

An electron microscope is a microscope that uses a beam of accelerated electrons as a source of illumination. As the wavelength of an electron can be up to 100,000 times shorter than that of visible light photons, electron microscopes have a higher resolving power than light microscopes and can reveal the structure of smaller objects. A scanning transmission electron microscope has achieved better than 50 pm resolution in annular dark-field imaging mode[1] and magnifications of up to about 10,000,000× whereas most light microscopes are limited by diffraction to about 200 nm resolution and useful magnifications below 2000×.

Electron microscopes have electron optical lens systems that are analogous to the glass lenses of an optical light microscope.

Electron microscopes are used to investigate the ultrastructure of a wide range of biological and inorganic specimens including microorganisms, cells, large molecules, biopsy samples, metals, and crystals. Industrially, electron microscopes are often used for quality control and failure analysis. Modern electron microscopes produce electron micrographs using specialized digital cameras and frame grabbers to capture the images.

Electron Microscope
A modern transmission electron microscope
Electron Microscope
Diagram of a transmission electron microscope
Ernst Ruska Electron Microscope - Deutsches Museum - Munich-edit
Electron microscope constructed by Ernst Ruska in 1933

History

Electron Interaction with Matter
Diagram illustrating the phenomena resulting from the interaction of highly energetic electrons with matter

In 1926 Hans Busch developed the electromagnetic lens.

According to Dennis Gabor, the physicist Leó Szilárd tried in 1928 to convince him to build an electron microscope, for which he had filed a patent.[2] The first prototype electron microscope, capable of four-hundred-power magnification, was developed in 1931 by the physicist Ernst Ruska and the electrical engineer Max Knoll.[3] The apparatus was the first practical demonstration of the principles of electron microscopy.[4] In May of the same year, Reinhold Rudenberg, the scientific director of Siemens-Schuckertwerke, obtained a patent for an electron microscope. In 1932, Ernst Lubcke of Siemens & Halske built and obtained images from a prototype electron microscope, applying the concepts described in Rudenberg's patent.[5]

In the following year, 1933, Ruska built the first electron microscope that exceeded the resolution attainable with an optical (light) microscope.[4] Four years later, in 1937, Siemens financed the work of Ernst Ruska and Bodo von Borries, and employed Helmut Ruska, Ernst's brother, to develop applications for the microscope, especially with biological specimens.[4][6] Also in 1937, Manfred von Ardenne pioneered the scanning electron microscope.[7] Siemens produced the first commercial electron microscope in 1938.[8] The first North American electron microscope was constructed in 1938, at the University of Toronto, by Eli Franklin Burton and students Cecil Hall, James Hillier, and Albert Prebus. Siemens produced a transmission electron microscope (TEM) in 1939.[9] Although current transmission electron microscopes are capable of two million-power magnification, as scientific instruments, they remain based upon Ruska’s prototype.

Types

Transmission electron microscope (TEM)

Operating principle of a transmission electron microscope

The original form of the electron microscope, the transmission electron microscope (TEM), uses a high voltage electron beam to illuminate the specimen and create an image. The electron beam is produced by an electron gun, commonly fitted with a tungsten filament cathode as the electron source. The electron beam is accelerated by an anode typically at +100 keV (40 to 400 keV) with respect to the cathode, focused by electrostatic and electromagnetic lenses, and transmitted through the specimen that is in part transparent to electrons and in part scatters them out of the beam. When it emerges from the specimen, the electron beam carries information about the structure of the specimen that is magnified by the objective lens system of the microscope. The spatial variation in this information (the "image") may be viewed by projecting the magnified electron image onto a fluorescent viewing screen coated with a phosphor or scintillator material such as zinc sulfide. Alternatively, the image can be photographically recorded by exposing a photographic film or plate directly to the electron beam, or a high-resolution phosphor may be coupled by means of a lens optical system or a fibre optic light-guide to the sensor of a digital camera. The image detected by the digital camera may be displayed on a monitor or computer.

The resolution of TEMs is limited primarily by spherical aberration, but a new generation of hardware correctors can reduce spherical aberration to increase the resolution in high-resolution transmission electron microscopy (HRTEM) to below 0.5 angstrom (50 picometres),[1] enabling magnifications above 50 million times.[10] The ability of HRTEM to determine the positions of atoms within materials is useful for nano-technologies research and development.[11]

Transmission electron microscopes are often used in electron diffraction mode. The advantages of electron diffraction over X-ray crystallography are that the specimen need not be a single crystal or even a polycrystalline powder, and also that the Fourier transform reconstruction of the object's magnified structure occurs physically and thus avoids the need for solving the phase problem faced by the X-ray crystallographers after obtaining their X-ray diffraction patterns.

One major disadvantage of the transmission electron microscope is the need for extremely thin sections of the specimens, typically about 100 nanometers. Creating these thin sections for biological and materials specimens is technically very challenging. Semiconductor thin sections can be made using a focused ion beam. Biological tissue specimens are chemically fixed, dehydrated and embedded in a polymer resin to stabilize them sufficiently to allow ultrathin sectioning. Sections of biological specimens, organic polymers, and similar materials may require staining with heavy atom labels in order to achieve the required image contrast.

Serial-section electron microscopy (ssEM)

One application of TEM is serial-section electron microscopy (ssEM), for example in analyzing the connectivity in volumetric samples of brain tissue by imaging many thin sections in sequence.[12]

Scanning electron microscope (SEM)

Operating principe of a scanning electron microscope
Bacillus subtilis image
Image of Bacillus subtilis taken with a 1960s electron microscope

The SEM produces images by probing the specimen with a focused electron beam that is scanned across a rectangular area of the specimen (raster scanning). When the electron beam interacts with the specimen, it loses energy by a variety of mechanisms. The lost energy is converted into alternative forms such as heat, emission of low-energy secondary electrons and high-energy backscattered electrons, light emission (cathodoluminescence) or X-ray emission, all of which provide signals carrying information about the properties of the specimen surface, such as its topography and composition. The image displayed by an SEM maps the varying intensity of any of these signals into the image in a position corresponding to the position of the beam on the specimen when the signal was generated. In the SEM image of an ant shown below and to the right, the image was constructed from signals produced by a secondary electron detector, the normal or conventional imaging mode in most SEMs.

Generally, the image resolution of an SEM is lower than that of a TEM. However, because the SEM images the surface of a sample rather than its interior, the electrons do not have to travel through the sample. This reduces the need for extensive sample preparation to thin the specimen to electron transparency. The SEM is able to image bulk samples that can fit on its stage and still be maneuvered, including a height less than the working distance being used, often 4 millimeters for high-resolution images. The SEM also has a great depth of field, and so can produce images that are good representations of the three-dimensional surface shape of the sample. Another advantage of SEMs comes with environmental scanning electron microscopes (ESEM) that can produce images of good quality and resolution with hydrated samples or in low, rather than high, vacuum or under chamber gases. This facilitates imaging unfixed biological samples that are unstable in the high vacuum of conventional electron microscopes.

Ant SEM
An image of an ant in a scanning electron microscope

Color

In their most common configurations, electron microscopes produce images with a single brightness value per pixel, with the results usually rendered in grayscale.[13] However, often these images are then colorized through the use of feature-detection software, or simply by hand-editing using a graphics editor. This may be done to clarify structure or for aesthetic effect and generally does not add new information about the specimen.[14]

In some configurations information about several specimen properties is gathered per pixel, usually by the use of multiple detectors.[15] In SEM, the attributes of topography and material contrast can be obtained by a pair of backscattered electron detectors and such attributes can be superimposed in a single color image by assigning a different primary color to each attribute.[16] Similarly, a combination of backscattered and secondary electron signals can be assigned to different colors and superimposed on a single color micrograph displaying simultaneously the properties of the specimen.[17]

Some types of detectors used in SEM have analytical capabilities, and can provide several items of data at each pixel. Examples are the Energy-dispersive X-ray spectroscopy (EDS) detectors used in elemental analysis and Cathodoluminescence microscope (CL) systems that analyse the intensity and spectrum of electron-induced luminescence in (for example) geological specimens. In SEM systems using these detectors, it is common to color code the signals and superimpose them in a single color image, so that differences in the distribution of the various components of the specimen can be seen clearly and compared. Optionally, the standard secondary electron image can be merged with the one or more compositional channels, so that the specimen's structure and composition can be compared. Such images can be made while maintaining the full integrity of the original signal, which is not modified in any way.

Reflection electron microscope (REM)

In the reflection electron microscope (REM) as in the TEM, an electron beam is incident on a surface but instead of using the transmission (TEM) or secondary electrons (SEM), the reflected beam of elastically scattered electrons is detected. This technique is typically coupled with reflection high energy electron diffraction (RHEED) and reflection high-energy loss spectroscopy (RHELS). Another variation is spin-polarized low-energy electron microscopy (SPLEEM), which is used for looking at the microstructure of magnetic domains.[18]

Scanning transmission electron microscope (STEM)

The STEM rasters a focused incident probe across a specimen that (as with the TEM) has been thinned to facilitate detection of electrons scattered through the specimen. The high resolution of the TEM is thus possible in STEM. The focusing action (and aberrations) occur before the electrons hit the specimen in the STEM, but afterward in the TEM. The STEMs use of SEM-like beam rastering simplifies annular dark-field imaging, and other analytical techniques, but also means that image data is acquired in serial rather than in parallel fashion. Often TEM can be equipped with the scanning option and then it can function both as TEM and STEM.

Sample preparation

Golden insect 01 Pengo
An insect coated in gold for viewing with a scanning electron microscope

Materials to be viewed under an electron microscope may require processing to produce a suitable sample. The technique required varies depending on the specimen and the analysis required:

  • Chemical fixation – for biological specimens aims to stabilize the specimen's mobile macromolecular structure by chemical crosslinking of proteins with aldehydes such as formaldehyde and glutaraldehyde, and lipids with osmium tetroxide.
  • Negative stain – suspensions containing nanoparticles or fine biological material (such as viruses and bacteria) are briefly mixed with a dilute solution of an electron-opaque solution such as ammonium molybdate, uranyl acetate (or formate), or phosphotungstic acid. This mixture is applied to a suitably coated EM grid, blotted, then allowed to dry. Viewing of this preparation in the TEM should be carried out without delay for best results. The method is important in microbiology for fast but crude morphological identification, but can also be used as the basis for high-resolution 3D reconstruction using EM tomography methodology when carbon films are used for support. Negative staining is also used for observation of nanoparticles.
  • Cryofixation – freezing a specimen so rapidly, in liquid ethane, and maintained at liquid nitrogen or even liquid helium temperatures, so that the water forms vitreous (non-crystalline) ice. This preserves the specimen in a snapshot of its solution state. An entire field called cryo-electron microscopy has branched from this technique. With the development of cryo-electron microscopy of vitreous sections (CEMOVIS), it is now possible to observe samples from virtually any biological specimen close to its native state.
  • Dehydration – or replacement of water with organic solvents such as ethanol or acetone, followed by critical point drying or infiltration with embedding resins. Also freeze drying.
  • Embedding, biological specimens – after dehydration, tissue for observation in the transmission electron microscope is embedded so it can be sectioned ready for viewing. To do this the tissue is passed through a 'transition solvent' such as propylene oxide (epoxypropane) or acetone and then infiltrated with an epoxy resin such as Araldite, Epon, or Durcupan;[19] tissues may also be embedded directly in water-miscible acrylic resin. After the resin has been polymerized (hardened) the sample is thin sectioned (ultrathin sections) and stained – it is then ready for viewing.
  • Embedding, materials – after embedding in resin, the specimen is usually ground and polished to a mirror-like finish using ultra-fine abrasives. The polishing process must be performed carefully to minimize scratches and other polishing artifacts that reduce image quality.
  • Metal shadowing – Metal (e.g. platinum) is evaporated from an overhead electrode and applied to the surface of a biological sample at an angle.[20] The surface topography results in variations in the thickness of the metal that are seen as variations in brightness and contrast in the electron microscope image.
  • Replication – A surface shadowed with metal (e.g. platinum, or a mixture of carbon and platinum) at an angle is coated with pure carbon evaporated from carbon electrodes at right angles to the surface. This is followed by removal of the specimen material (e.g. in an acid bath, using enzymes or by mechanical separation[21]) to produce a surface replica that records the surface ultrastructure and can be examined using transmission electron microscopy.
  • Sectioning – produces thin slices of the specimen, semitransparent to electrons. These can be cut on an ultramicrotome with a glass or diamond knife to produce ultra-thin sections about 60–90 nm thick. Disposable glass knives are also used because they can be made in the lab and are much cheaper.
  • Staining – uses heavy metals such as lead, uranium or tungsten to scatter imaging electrons and thus give contrast between different structures, since many (especially biological) materials are nearly "transparent" to electrons (weak phase objects). In biology, specimens can be stained "en bloc" before embedding and also later after sectioning. Typically thin sections are stained for several minutes with an aqueous or alcoholic solution of uranyl acetate followed by aqueous lead citrate.[22]
  • Freeze-fracture or freeze-etch – a preparation method[23][24] particularly useful for examining lipid membranes and their incorporated proteins in "face on" view.[25] The fresh tissue or cell suspension is frozen rapidly (cryofixation), then fractured by breaking[26] or by using a microtome while maintained at liquid nitrogen temperature. The cold fractured surface (sometimes "etched" by increasing the temperature to about −100 °C for several minutes to let some ice sublime) is then shadowed with evaporated platinum or gold at an average angle of 45° in a high vacuum evaporator. The second coat of carbon, evaporated perpendicular to the average surface plane is often performed to improve the stability of the replica coating. The specimen is returned to room temperature and pressure, then the extremely fragile "pre-shadowed" metal replica of the fracture surface is released from the underlying biological material by careful chemical digestion with acids, hypochlorite solution or SDS detergent. The still-floating replica is thoroughly washed free from residual chemicals, carefully fished up on fine grids, dried then viewed in the TEM.
  • Freeze-fracture replica immunogold labeling (FRIL) – the freeze-fracture method has been modified to allow the identification of the components of the fracture face by immunogold labeling. Instead of removing all the underlying tissue of the thawed replica as the final step before viewing in the microscope the tissue thickness is minimized during or after the fracture process. The thin layer of tissue remains bound to the metal replica so it can be immunogold labeled with antibodies to the structures of choice. The thin layer of the original specimen on the replica with gold attached allows the identification of structures in the fracture plane.[27] There are also related methods which label the surface of etched cells[28] and other replica labeling variations.[29]
  • Ion beam milling – thins samples until they are transparent to electrons by firing ions (typically argon) at the surface from an angle and sputtering material from the surface. A subclass of this is focused ion beam milling, where gallium ions are used to produce an electron transparent membrane in a specific region of the sample, for example through a device within a microprocessor. Ion beam milling may also be used for cross-section polishing prior to SEM analysis of materials that are difficult to prepare using mechanical polishing.
  • Conductive coating – an ultrathin coating of electrically conducting material, deposited either by high vacuum evaporation or by low vacuum sputter coating of the sample. This is done to prevent the accumulation of static electric fields at the specimen due to the electron irradiation required during imaging. The coating materials include gold, gold/palladium, platinum, tungsten, graphite, etc.
  • Earthing – to avoid electrical charge accumulation on a conductively coated sample, it is usually electrically connected to the metal sample holder. Often an electrically conductive adhesive is used for this purpose.

Disadvantages

Electron microscopes are expensive to build and maintain, but the capital and running costs of confocal light microscope systems now overlaps with those of basic electron microscopes. Microscopes designed to achieve high resolutions must be housed in stable buildings (sometimes underground) with special services such as magnetic field canceling systems.

The samples largely have to be viewed in vacuum, as the molecules that make up air would scatter the electrons. An exception is liquid-phase electron microscopy [30] using either a closed liquid cell or an environmental chamber, for example, in the environmental scanning electron microscope, which allows hydrated samples to be viewed in a low-pressure (up to 20 Torr or 2.7 kPa) wet environment. Various techniques for in situ electron microscopy of gaseous samples have been developed as well.[31]

Scanning electron microscopes operating in conventional high-vacuum mode usually image conductive specimens; therefore non-conductive materials require conductive coating (gold/palladium alloy, carbon, osmium, etc.). The low-voltage mode of modern microscopes makes possible the observation of non-conductive specimens without coating. Non-conductive materials can be imaged also by a variable pressure (or environmental) scanning electron microscope.

Small, stable specimens such as carbon nanotubes, diatom frustules and small mineral crystals (asbestos fibres, for example) require no special treatment before being examined in the electron microscope. Samples of hydrated materials, including almost all biological specimens have to be prepared in various ways to stabilize them, reduce their thickness (ultrathin sectioning) and increase their electron optical contrast (staining). These processes may result in artifacts, but these can usually be identified by comparing the results obtained by using radically different specimen preparation methods. Since the 1980s, analysis of cryofixed, vitrified specimens has also become increasingly used by scientists, further confirming the validity of this technique.[32][33][34]

Applications

Semiconductor and data storage

Biology and life sciences

Materials research
Industry

See also

References

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External links

General

History

Other

Basal lamina

The basal lamina is a layer of extracellular matrix secreted by the epithelial cells, on which the epithelium sits. It is often incorrectly referred to as the basement membrane, though it does constitute a portion of the basement membrane. The basal lamina is visible only with the electron microscope, where it appears as an electron-dense layer that is 20–100 nm thick (with some exceptions that are thicker, such as basal lamina in lung alveoli and renal glomeruli).

Cathodoluminescence

Cathodoluminescence is an optical and electromagnetic phenomenon in which electrons impacting on a luminescent material such as a phosphor, cause the emission of photons which may have wavelengths in the visible spectrum. A familiar example is the generation of light by an electron beam scanning the phosphor-coated inner surface of the screen of a television that uses a cathode ray tube. Cathodoluminescence is the inverse of the photoelectric effect, in which electron emission is induced by irradiation with photons.

Dark-field microscopy

Dark-field microscopy (also called dark-ground microscopy) describes microscopy methods, in both light and electron microscopy, which exclude the unscattered beam from the image. As a result, the field around the specimen (i.e., where there is no specimen to scatter the beam) is generally dark.

Electron backscatter diffraction

Electron backscatter diffraction (EBSD) is a Scanning Electron Microscope based microstructural-crystallographic characterisation technique commonly used in the study of crystalline or polycrystalline materials. The technique can provide information about the structure, crystal orientation, phase or strain in the material. Traditionally these types of studies have been carried out using X-ray diffraction (XRD), neutron diffraction and/or electron diffraction in a Transmission electron microscope.

Environmental scanning electron microscope

The environmental scanning electron microscope or ESEM is a scanning electron microscope (SEM) that allows for the option of collecting electron micrographs of specimens that are "wet," uncoated, or both by allowing for a gaseous environment in the specimen chamber. Although there were earlier successes at viewing wet specimens in internal chambers in modified SEMs, the ESEM with its specialized electron detectors (rather than the standard Everhart-Thornley detector) and its differential pumping systems, to allow for the transfer of the electron beam from the high vacuums in the gun area to the high pressures attainable in its specimen chamber, make it a complete and unique instrument designed for the purpose of imaging specimens in their natural state. The instrument was designed originally by Gerasimos Danilatos while working at the University of New South Wales.

Ernst Ruska

Ernst August Friedrich Ruska (25 December 1906 – 27 May 1988) was a German physicist who won the Nobel Prize in Physics in 1986 for his work in electron optics, including the design of the first electron microscope.

JEOL

JEOL, Ltd. (日本電子, Nihon Denshi Kabushiki-gaisha, Nihon meaning Japan and Denshi meaning electron) is a major developer and manufacturer of electron microscopes and other scientific instruments, industrial equipment and medical equipment.Its headquarters are in Tokyo, Japan, with 25 domestic and foreign subsidiaries and associated companies as of 2014. It is listed in the top ten businesses worldwide for analytical laboratory instrument manufacturing. JEOL's instruments are used by researchers around the world, including the University of Cambridge, University of Oxford, and MIT.It has been included in the Activest Lux Nanotech Mutual Fund and the WestLB Nanotech Fund.

James Hillier

James Hillier, (August 22, 1915 – January 15, 2007) was a Canadian-American scientist and inventor who designed and built, with Albert Prebus, the first successful high-resolution electron microscope in North America in 1938.

James Menter

Sir James Woodham Menter, (22 August 1921 – 18 July 2006) was a British physicist.

He was born in Teynham, Kent and was educated at the Dover Grammar School for Boys, where he won a scholarship to study Natural Sciences at Peterhouse, Cambridge. His studies were interrupted by the Second World War, during which he was engaged on trials of Under Water Sound Detection systems at the Admiralty Research Station in Fairlie, Ayrshire. He completed his degree in 1945 and did a PhD on the subject of the use of the electron microscope to examine the micro-topography of surfaces.In 1961 he went to work at the Tube Investments Research Laboratory at Hinxton Hall, Cambridgeshire. There he was able to acquire the latest and most powerful Electron Microscope then available, the Siemens Elmiscop 1, and soon demonstrated its potential to determine the atomic structure of crystalline solids by resolving the structure of platinum phthalocyanine. In 1965 he was appointed Director of Research and Development at the establishment and in 1968 made a member of the main board of the company.

He was elected a Fellow of the Royal Society in 1966, becoming Vice-President and Treasurer of the society. He was also President of the newly renamed Institute of Physics for 1970–1972 and knighted in 1973. In 1974 he was made an Honorary Fellow of the Royal Microscopical Society.

He left TI in 1976 to become Principal of Queen Mary College, London, holding the position until 1986.

He died in Aberfeldy, Perthshire in 2006. He had married Jean Whyte-Smith and had two sons and a daughter.

Low-voltage electron microscope

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

Low voltage imaging in transmitted electrons is possible in many new scanning electron detector.

Low cost alternative is dedicated table top low voltage transmission electron microscope. While its architecture is very similar to a conventional transmission electron microscope, it has a few key changes that enable it to take advantage of a 5 keV electron source, but trading off many advantages of higher voltage operations, including higher resolution, possibility of X-ray microanalysis and EELS, etc... Recently a new low voltage transmission electron microscope has been introduced that operates at variable voltage ranges between 6–25 kV.

Microscope

A microscope (from the Ancient Greek: μικρός, mikrós, "small" and σκοπεῖν, skopeîn, "to look" or "see") is an instrument used to see objects that are too small to be seen by the naked eye. Microscopy is the science of investigating small objects and structures using such an instrument. Microscopic means invisible to the eye unless aided by a microscope.

There are many types of microscopes, and they may be grouped in different ways. One way is to describe the way the instruments interact with a sample to create images, either by sending a beam of light or electrons to a sample in its optical path, or by scanning across, and a short distance from the surface of a sample using a probe. The most common microscope (and the first to be invented) is the optical microscope, which uses light to pass through a sample to produce an image. Other major types of microscopes are the fluorescence microscope, the electron microscope (both the transmission electron microscope and the scanning electron microscope) and the various types of scanning probe microscopes.

Phenom

Phenom may refer to:

Phenom (TV series), an American sitcom

AMD Phenom, the 64-bit AMD desktop processor line based on the K10 microarchitecture

Phenom II, a family of AMD's multi-core 45 nm processors using the AMD K10 microarchitecture

Embraer Phenom 100, a very light jet

Phenom (electron microscope), a fast electron microscope

Phenom (rock group), a progressive rock group from Bangalore, India

"Phenom" (song), a song by American rapper Xzibit

The Phenom, a 2015 sports drama film starring Ethan Hawke

The Undertaker (born 1965; also "The Phenom"), American professional wrestler

Vitor Belfort (born 1977; also "The Phenom"), Brazilian mixed martial artist

Reinhold Rudenberg

Reinhold Rudenberg (or Rüdenberg; February 4, 1883 – December 25, 1961) was a German-American electrical engineer and inventor, credited with many innovations in the electric power and related fields. Aside from improvements in electric power equipment, especially large alternating current generators, among others were the electrostatic-lens electron microscope, carrier-current communications on power lines, a form of phased array radar, an explanation of power blackouts, preferred number series, and the number prefix "Giga-".

Scanning confocal electron microscopy

Scanning confocal electron microscopy (SCEM) is an electron microscopy technique analogous to scanning confocal optical microscopy (SCOM). In this technique, the studied sample is illuminated by a focussed electron beam, as in other scanning microscopy techniques, such as scanning transmission electron microscopy or scanning electron microscopy. However, in SCEM, the collection optics is arranged symmetrically to the illumination optics to gather only the electrons that pass the beam focus. This results in superior depth resolution of the imaging. The technique is relatively new and is being actively developed.

Scanning electron microscope

A scanning electron microscope (SEM) is a type of electron microscope that produces images of a sample by scanning the surface with a focused beam of electrons. The electrons interact with atoms in the sample, producing various signals that contain information about the surface topography and composition of the sample. The electron beam is scanned in a raster scan pattern, and the position of the beam is combined with the intensity of the detected signal to produce an image. In the most common SEM mode, secondary electrons emitted by atoms excited by the electron beam are detected using an Everhart-Thornley detector. The number of secondary electrons that can be detected, and thus the signal intensity, depends, among other things, on specimen topography. SEM can achieve resolution better than 1 nanometer.

Specimens are observed in high vacuum in conventional SEM, or in low vacuum or wet conditions in variable pressure or environmental SEM, and at a wide range of cryogenic or elevated temperatures with specialized instruments.

Scanning transmission electron microscopy

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 (with the typical spot size 0.05 – 0.2 nm) which is then scanned over the sample in a raster illumination system constructed in a way that at each point sample illuminated 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.

A typical STEM is a conventional transmission electron microscope equipped with additional scanning coils, detectors and necessary circuitry, which allows it to switch between operating as a STEM, or a CTEM; however, dedicated STEMs are also manufactured.

High resolution scanning transmission electron microscopes require exceptionally stable room environments. In order to obtain atomic resolution images in STEM, the level of vibration, temperature fluctuations, electromagnetic waves, and acoustic waves must be limited in the room housing the microscope.

Thomas Eugene Everhart

Thomas Eugene Everhart FREng (born February 15, 1932, Kansas City, Missouri) is an American educator and physicist. His area of expertise is the physics of electron beams. Together with Richard F. M. Thornley he designed the Everhart-Thornley detector. These detectors are still in use in scanning electron microscopes, even though the first such detector was made available as early as 1956.

Everhart was elected to the National Academy of Engineering in 1978. He was appointed an International Fellow of the Royal Academy of Engineering in 1990. He served as Chancellor of the University of Illinois at Urbana-Champaign from 1984 to 1987 and as the President of the California Institute of Technology from 1987 to 1997.

Transmission electron microscopy

Transmission electron microscopy (TEM, an abbreviation which can also stand for the instrument, a transmission electron microscope) 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 sensor such as a scintillator attached to a charge-coupled device.

Transmission electron microscopes are capable of imaging at a significantly higher resolution than light microscopes, owing to the smaller de Broglie wavelength of electrons. This enables the instrument to capture fine detail—even as small as a single column of atoms, which is thousands of times smaller than a resolvable object seen in a light microscope. Transmission electron microscopy is a major analytical method in the physical, chemical and biological sciences. TEMs find application in cancer research, virology, and materials science as well as pollution, nanotechnology and semiconductor research.

TEM instruments boast an enormous array of operating modes including conventional imaging, scanning TEM imaging (STEM), diffraction, spectroscopy, and combinations of these. Even within conventional imaging, there are many fundamentally different ways that contrast is produced, called "image contrast mechanisms." Contrast can arise from position-to-position differences in the thickness or density ("mass-thickness contrast"), atomic number ("Z contrast," referring to the common abbreviation Z for atomic number), crystal structure or orientation ("crystallographic contrast" or "diffraction contrast"), the slight quantum-mechanical phase shifts that individual atoms produce in electrons that pass through them ("phase contrast"), the energy lost by electrons on passing through the sample ("spectrum imaging") and more. Each mechanism tells the user a different kind of information, depending not only on the contrast mechanism but on how the microscope is used—the settings of lenses, apertures, and detectors. What this means is that a TEM is capable of returning an extraordinary variety of nanometer- and atomic-resolution information, in ideal cases revealing not only where all the atoms are but what kinds of atoms they are and how they are bonded to each other. For this reason TEM is regarded as an essential tool for nanoscience in both biological and materials fields.

The first TEM was demonstrated by Max Knoll and Ernst Ruska in 1931, with this group developing the first TEM with resolution greater than that of light in 1933 and the first commercial TEM in 1939. In 1986, Ruska was awarded the Nobel Prize in physics for the development of transmission electron microscopy.

Ultrastructure

Ultrastructure (or ultra-structure) is the architecture of cells and biomaterials that is visible at higher magnifications than found on a standard optical light microscope. This traditionally meant the resolution and magnification range of a conventional transmission electron microscope (TEM) when viewing biological specimens such as cells, tissue, or organs. Ultrastructure can also be viewed with scanning electron microscopy and super-resolution microscopy, although TEM is a standard histology technique for viewing ultrastructure. Such cellular structures as organelles, which allow the cell to function properly within its specified environment, can be examined at the ultrastructural level.

Ultrastructure, along with molecular phylogeny, is a reliable phylogenetic way of classifying organisms. Features of ultrastructure are used industrially to control material properties and promote biocompatibility.

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