Diagram of a transmission electron microscope

A 1973 Siemens electron microscope, Musée des Arts et Métiers, Paris

An electron microscope is a type of microscope that uses a beam of electrons to illuminate the specimen and produce a magnified image. Electron microscopes (EM) have a greater resolving power than a light-powered optical microscope, because electrons have wavelengths about 100,000 times shorter than visible light (photons), and can achieve better than 50 pm resolution^[1] and magnifications of up to about 10,000,000x, whereas ordinary, non-confocal light microscopes are limited by diffraction to about 200 nm resolution and useful magnifications below 2000x.

The electron microscope uses electrostatic and electromagnetic "lenses" to control the electron beam and focus it to form an image. These lenses are analogous to, but different from the glass lenses of an optical microscope that form a magnified image by focusing light on or through the specimen. Electron microscopes are used to observe a wide range of biological and inorganic specimens including microorganisms, cells, large molecules, biopsy samples, metals, and crystals. Industrially, the electron microscope is often used for quality control and failure analysis.

History[link]

Electron microscope constructed by Ernst Ruska in 1933

The electron microscope was invented and patented by Hungarian physicist Leó Szilárd who declined to construct it.^[2] Instead, German physicist Ernst Ruska and electrical engineer Max Knoll constructed the prototype electron microscope in 1931, capable of four-hundred-power magnification; the apparatus was a practical application of the principles of electron microscopy.^[3] Two years later, in 1933, Ruska built an electron microscope that exceeded the resolution attainable with an optical (lens) microscope.^[3] Moreover, Reinhold Rudenberg, the scientific director of Siemens-Schuckertwerke, obtained the patent for the electron microscope in May 1931. Family illness compelled the electrical engineer to devise an electrostatic microscope, because he wanted to make visible the poliomyelitis virus.

In 1932, Ernst Lubcke of Siemens & Halske built and obtained images from a prototype electron microscope, applying concepts described in the Rudenberg patent applications.^[4] Five years later (1937), the firm 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 biologic specimens.^[3][5] Also in 1937, Manfred von Ardenne pioneered the scanning electron microscope.^[6] The first practical electron microscope was constructed in 1938, at the University of Toronto, by Eli Franklin Burton and students Cecil Hall, James Hillier, and Albert Prebus; and Siemens produced the first commercial transmission electron microscope (TEM) in 1939.^[7] Although contemporary electron microscopes are capable of two million-power magnification, as scientific instruments, they remain based upon Ruska’s prototype.

Types[link]

Transmission electron microscope (TEM)[link]

The original form of electron microscope, the transmission electron microscope (TEM) uses a high voltage electron beam to create an image. The electrons are emitted 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 CCD (charge-coupled device) camera. The image detected by the CCD may be displayed on a monitor or computer.

Resolution of the TEM is limited primarily by spherical aberration, but a new generation of aberration correctors have been able to partially overcome spherical aberration to increase resolution. Hardware correction of spherical aberration for the high-resolution transmission electron microscopy (HRTEM) has allowed the production of images with resolution below 0.5 angstrom (50 picometres)^[1] and magnifications above 50 million times.^[8] The ability to determine the positions of atoms within materials has made the HRTEM an important tool for nano-technologies research and development.^[9]

An important mode of TEM utilization is electron diffraction. 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 of a single crystal or polycrystalline powder. The major disadvantage of the transmission electron microscope is the need for extremely thin sections of the specimens, typically about 100 nanometers. Biological specimens typically require to be 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 special `staining' with heavy atom labels in order to achieve the required image contrast.

Scanning electron microscope[link]

An image of an ant in a scanning electron microscope

Unlike the TEM, where electrons of the high voltage beam carry the image of the specimen, the electron beam of the scanning electron microscope (SEM)^[10] does not at any time carry a complete image of the specimen. 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, 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 at 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 about an order of magnitude poorer than that of a TEM. However, because the SEM image relies on surface processes rather than transmission, it is able to image bulk samples up to many centimetres in size and (depending on instrument design and settings) has a great depth of field, and so can produce images that are good representations of the three-dimensional shape of the sample. Another advantage of SEM is its variety called environmental scanning electron microscope (ESEM) can produce images of sufficient quality and resolution with the samples being wet or contained in low vacuum or gas. This greatly facilitates imaging biological samples that are unstable in the high vacuum of conventional electron microscopes.

Reflection electron microscope[link]

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.^[11]

Scanning transmission electron microscope[link]

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.

Low-voltage electron microscope[link]

The low-voltage electron microscope (LVEM) is a combination of SEM, TEM and STEM in one instrument, which operates at relatively low electron accelerating voltage of 5 kV. Low voltage reduces the specimen damage by the incident electrons and increases image contrast that is especially important for biological specimens. This increase in contrast significantly reduces, or even eliminates the need to stain. Sectioned samples generally need to be thinner than they would be for conventional TEM (20–65 nm). Resolutions of a few nm are possible in TEM, SEM and STEM modes.^[12][13]

Sample preparation[link]

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.
• Cryofixation – freezing a specimen so rapidly, to liquid nitrogen or even liquid helium temperatures, 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.^{[citation needed]}
• Dehydration – freeze drying, or replacement of water with organic solvents such as ethanol or acetone, followed by critical point drying or infiltration with embedding resins.
• 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) and then infiltrated with an epoxy resin such as Araldite, Epon, or Durcupan;^[14] tissues may also be embedded directly in water-miscible acrylic resin. After the resin has been polymerised (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.
• Sectioning – produces thin slices of specimen, semitransparent to electrons. These can be cut on an ultramicrotome with a diamond knife to produce ultra-thin slices 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.
• Freeze-fracture or freeze-etch – a preparation method particularly useful for examining lipid membranes and their incorporated proteins in "face on" view. The fresh tissue or cell suspension is frozen rapidly (cryofixation), then fractured by simply breaking 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. A second coat of carbon, evaporated perpendicular to the average surface plane is often performed to improve 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.
• 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.

Disadvantages[link]

False-color SEM image of the filter setae of an Antarctic krill. (Raw electron microscope images carry no color information.)
Pictured: First degree filter setae with a V pattern of second degree setae pointing towards the inside of the feeding basket. The purple ball is 1 µm in diameter.

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 cancelling systems.

The samples largely have to be viewed in vacuum, as the molecules that make up air would scatter the electrons. One exception is the environmental scanning electron microscope, which allows hydrated samples to be viewed in a low-pressure (up to 20 Torr/2.7 kPa) and/or wet environment.

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.) Low-voltage mode of modern microscopes makes possible 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. It is generally believed by scientists working in the field that as results from various preparation techniques have been compared and that there is no reason that they should all produce similar artifacts, it is reasonable to believe that electron microscopy features correspond with those of living cells. Since the 1980s, analysis of cryofixed, vitrified specimens has also become increasingly used by scientists, further confirming the validity of this technique.^[15][16][17]

Applications[link]

See also[link]

References[link]

External links[link]

Wikimedia Commons has media related to: Electron microscope images

• Science Aid: Electron Microscopy High School (GCSE, A Level) resource
• Cell Centered Database – Electron microscopy data

General[link]

• Nanohedron.com|Nano image gallery beautiful images generated with electron microscopes.
• electron microscopy Website of the ETH Zurich: Very good graphics and images, which illustrate various procedures.
• Environmental Scanning Electron Microscope (ESEM)
• X-ray element analysis in electron microscope – Information portal with X-ray microanalysis and EDX contents
• Introduction to Electron Microscopy by Eva Nogales (UC Berkeley)
• good introduction to electron microscopy by David Szondy

History[link]

• John H.L. Watson: Very early Electron Microscopy in the Department of Physics, the University of Toronto – A personal recollection
• Rubin Borasky Electron Microscopy Collection, 1930–1988 Archives Center, National Museum of American History, Smithsonian Institution.

Other[link]

• The Royal Microscopical Society, Electron Microscopy Section (UK)
• Albert Lleal. Natural history subjects at Scanning Electron Microscope SEM

Dr. Eva Nogales (b. Madrid, Spain) is a biophysicist at the Lawrence Berkeley National Laboratory. She is a Professor of Biochemistry and Molecular Biology at the University of California, Berkeley and a Howard Hughes Medical Institute investigator.

Eva Nogales works on structural and functional characterization of macromolecular complexes. Her lab uses electron microscopy, computational image analysis as well as functional biochemical assays to gain insights into function and regulation of the large biological assemblies.

Biography

Eva Nogales obtained her BS degree in physics from the Autonomous University of Madrid in 1988. She earned her PhD from the University of Keele in 1992 while working at the Synchrotron Radiation Source under the supervision of Joan Bordas.

During her post-doctoral work in the Ken Downing lab, Eva Nogales was the first to determine the atomic structure of tubulin by electron crystallography.^[1] She now continues to study microtubule dynamics in her own laboratory.^[2] In addition to microtubules, Dr. Nogales explores the structural and functional aspects of large macromolecular assemblies such as eukaryotic transcription and translation initiation complexes.

Awards and honors

Dr. Nogales is a recipient of several awards including Early Career Award by the American Society for Cell Biology (2005), and the Chabot Science Award for Excellence (2006).

Personal information

Eva Nogales is married to Howard Padmore and they have two children.

This article about a Spanish scientist is a stub. You can help Wikipedia by expanding it. v t e

This article about a biologist is a stub. You can help Wikipedia by expanding it. v t e

Electron microscope constructed by Ernst Ruska in 1933

Ernst August Friedrich Ruska (25 December 1906 – 27 May 1988^[1]) 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.

Ruska was born in Heidelberg. He was educated at the Technical University of Munich from 1925 to 1927 and then entered the Technical University of Berlin, where he posited that microscopes using electrons, with wavelengths 1000 times shorter than those of light, could provide a more detailed picture of an object than a microscope utilizing light, in which magnification is limited by the size of the wavelengths. In 1931, he demonstrated that a magnetic coil could act as an electron lens, and used several coils in a series to build the first electron microscope in 1933.

After completing his PhD in 1933, Ruska continued to work in the field of electron optics, first at Fernseh Ltd in Berlin-Zehlendorf, and then from 1937 at Siemens-Reiniger-Werke AG. At Siemens, he was involved in developing the first commercially-produced electron microscope in 1939. As well as developing the technology of electron microscopy while at Siemens, Ruska also worked at other scientific institutions, and encouraged Siemens to set up a laboratory for visiting researchers, which was initially headed by Ruska's brother Helmut, a medical doctor who developed the use of the electron microscope for medical and biological applications.

After leaving Siemens in 1955, Ruska served as director of the Institute for Electron Microscopy of the Fritz Haber Institute until 1974. Concurrently, he served at the institute and as professor at the Technical University of Berlin from 1957 until his retirement in 1974.

In 1986, he was awarded half of the Nobel Prize in Physics for his many achievements in electron optics; Gerd Binnig and Heinrich Rohrer won a quarter each for their design of the scanning tunneling microscope. He died in West Berlin in 1988.

References[link]

• Nobel Prize press release
• Ernst Ruska's official Nobel autobiography
• Website containing memorial information published by the Ruska family