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electron microscope

 
Dictionary: electron microscope
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n.
Any of a class of microscopes that use electrons rather than visible light to produce magnified images, especially of objects having dimensions smaller than the wavelengths of visible light, with linear magnification approaching or exceeding a million (106).


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Sci-Tech Encyclopedia: Electron microscope
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A device for forming greatly magnified images of objects by means of electrons. Electron microscopes serve primarily two purposes: the visual examination of structures too fine to be resolved with ordinary, or light, microscopes, and the study of surfaces that emit electrons. The first function made transmission electron microscopes essential research tools in biology, chemistry, and metallurgy. Beginning in the 1960s the scanning electron microscope came to play an increasingly important role in the study of the surfaces of solid objects at more moderate magnifications. Various emission electron microscopes serve more specialized research purposes. See also Field-emission microscopy; Scanning electron microscope.

A transmission electron microscope consists in its simplest form of a source supplying a beam of electrons of uniform velocity, a condenser lens for concentrating the electrons on the specimen, a specimen stage for displacing the specimen which transmits the electron beam, an objective lens, a projector lens, and a fluorescent screen on which the final image is observed. For permanent record of the image, the fluorescent screen is replaced by a photographic plate or film.

Electrons are strongly scattered by all forms of matter, including air. Hence the entire instrument must be evacuated to about 10−4 mmHg (10−7 atm or 10−2 pascal). Furthermore, the lenses cannot be material in nature. Instead, they are electric or magnetic fields, symmetrical about the axis of the instrument, that have the property of bending the electron paths toward the axis, just as converging glass lenses bend light rays toward their axis. Lens strength is varied by varying the current. Most electron microscopes employ magnetic lenses of this type. These have yielded the highest resolution and magnification attained.

However, good results have also been obtained with electron microscopes employing unipotential electrostatic lenses and magnetic lenses excited by permanent magnets. See also Electron lens; Electrostatic lens; Magnetic lens.

A microscope can, at best, permit the discrimination of two point objects greater than 0.7λ/sin θ apart. Here λ is the wavelength of the illuminating radiation, and θ is the aperture angle of the cone of radiation that participates in forming the image. For green light, λ = 500 nanometers. Even for ultraviolet radiation in an immersion medium of refractive index 1.5, λ is no less than 170 nm. Since light and ultraviolet microscope objectives can be designed to utilize practically all the radiation passing through the specimen, sin θ ≅ 1, and the least resolvable distance for the ultraviolet microscope is about 100 nm. For 50- to 100-kV electrons, such as are commonly employed in electron microscopes, the wavelength range is 0.0053–0.0037 nm. Hence, even though a cone of radiation with an aperture angle less than 0.01 radian contributes to an image of optimum sharpness, object separations smaller than 0.3 nm have been resolved with the electron microscope. Thus the electron microscope has several hundred times the resolving power of the light microscope. Similarly, whereas the maximum useful magnification of the light microscope is about 2000, that of the electron microscope may approach 1,000,000. The maximum useful magnification is the least magnification of the image that reveals to the observer all the specimen detail that the microscope is capable of conveying.

Electrons are commonly emitted from the tip of a fine tungsten-wire hairpin filament or, to further reduce the size of the effective electron source, from a sharply pointed segment of wire welded to the filament tip. The filament is maintained at a carefully stabilized negative potential of 50–100 kV with respect to the remainder of the instrument. Electrons enter the instrument through an anode aperture. The intensity and convergence of the electron beam that is falling on the specimen are adjusted by varying the coil current of the condenser lens. Image contrasts are formed by the scattering of electrons out of the narrow cone that contributes to the formation of the image; denser or thicker portions of the specimen scatter more electrons and hence appear darker in the image. The sharpness of the image observed on the screen is adjusted by varying the objective coil current, and its magnification by varying the projector coil current. Both currents must be carefully stabilized to yield high resolution.

In addition to the standard transmission microscopes operating at 50–100 kV, a number of very high-voltage instruments have been constructed (for operation up to 1500 kV). The advantage of high-voltage electron microscopy does not lie in greater resolving power but in increased penetration, which is particularly valuable in the direct study of metal sections prepared with a microtome.

The application of the electron miscroscope to examination and investigation of the ultramicrostructure of materials has become so extensive that there is hardly an area in biological and nonbiological research where electron microscopy does not play a role. In biological and medical research the development of sectioning techniques has extended electron microscopy down to observations at the macromolecular level for delineation of the complex organization of cell components such as membranes, mitochondria, endoplasmic reticulum, and ribosomes. Other techniques have been developed for studies on the structure of virus and even individual proteins and nucleic acids. Nonbiological solid materials have also become objects of extensive and fruitful investigation. Diffraction microscopy, together with the development of adequate techniques of sectioning and thinning crystalline materials such as metals, now makes the observation of defect structure in solids an important aspect of electron microscopy. See also Electron diffraction.

Dental Dictionary: electron microscope
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n

A microsope in which electron beams with wavelengths shorter than those of visible light are used in place of visible light, allowing much greater resolution and magnification of the object.

Science Dictionary: electron microscope
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A device that uses electrons instead of light to form images of very small objects, such as individual parts of small living things.

Wikipedia: Electron microscope
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Diagram of a transmission electron microscope

An electron microscope is a type of microscope, a scientific instrument which is used to magnify things on a fine scale. Electron microscopes use a particle beam of electrons to illuminate a specimen and create a highly-magnified image. They have much greater resolving power than light microscopes that use electromagnetic radiation and can obtain much higher magnifications of up to 1 million times, while the best light microscopes are limited to magnifications of 1000 times. Both electron and light microscopes have resolution limitations, imposed by the wavelength of the radiation they use. The greater resolution and magnification of the electron microscope is because the de Broglie wavelength of an electron is much smaller than that of a photon of visible light. The electron microscope uses electrostatic and electromagnetic lenses in forming the image by controlling the electron beam to focus it at a specific plane relative to the specimen.
This manner is similar to how a light microscope uses glass lenses to focus light on or through a specimen to form an image.

Contents

History

Electron microscope constructed by Ernst Ruska in 1933

The first electron microscope prototype was built in 1931 by the German engineers Ernst Ruska and Max Knoll.[1] Although this initial instrument was capable of magnifying objects by only four hundred times, it demonstrated the principles of an electron microscope. Two years later, Ruska constructed an electron microscope that exceeded the resolution possible with an optical microscope.[1]

Reinhold Rudenberg, the scientific director of Siemens, had patented the electron microscope in 1931, stimulated by family illness to make the poliomyelitis virus particle visible. In 1937 Siemens began funding Ruska and Bodo von Borries to develop an electron microscope. Siemens also employed Ruska's brother Helmut to work on applications, particularly with biological specimens.[2][3]

In the same decade Manfred von Ardenne pioneered the scanning electron microscope and his universal electron microscope.[4]

Siemens produced the first commercial Transmission Electron Microscope (TEM) in 1939, but the first practical electron microscope had been built at the University of Toronto in 1938, by Eli Franklin Burton and students Cecil Hall, James Hillier, and Albert Prebus.[5]

Although modern electron microscopes can magnify objects up to two million times, they are still based upon Ruska's prototype. The electron microscope is an essential item of equipment in many laboratories. Researchers use them to examine biological materials (such as microorganisms and cells), a variety of large molecules, medical biopsy samples, metals and crystalline structures and the characteristics of various surfaces. The electron microscope is also used extensively for inspection, quality assurance and failure analysis applications in industry, including, in particular, semiconductor device fabrication.

Types

Transmission electron microscope (TEM)

Main article: Transmission electron microscope

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") is viewed by projecting the magnified electron image onto a fluorescent viewing screen coated with a phosphor or scintillator material such as zinc sulfide. 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 TEM (HRTEM) has allowed the production of images with resolution below 0.5 Ångström (50 picometres)[6] at magnifications above 50 million times.[7] The ability to determine the positions of atoms within materials has made the HRTEM an important tool for nano-technologies research and development.[8]

Scanning electron microscope (SEM)

Main article: Scanning electron microscope
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)[9] 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). At each point on the specimen the incident electron beam loses some energy, and that lost energy is converted into other forms, such as heat, emission of low-energy secondary electrons, light emission (cathodoluminescence) or x-ray emission. The display of the 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.

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 spectrum (RHELS). Another variation is Spin-Polarized Low-Energy Electron Microscopy (SPLEEM), which is used for looking at the microstructure of magnetic domains.[10]

Scanning transmission electron microscope (STEM)

Main article: Scanning transmission electron microscopy

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.

Low voltage electron microscope (LVEM)

The low voltage electron microscope (LVEM) is a combination of SEM, TEM and STEM in one instrument, which operated at relatively low electron accelerating voltage of 5 kV. Low voltage increases image contrast which 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 (20-65nm). Resolutions of a few nm are possible in TEM, SEM and STEM modes. [11][12]

Sample preparation

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.
  • 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 epoxy propane and then infiltrated with a resin such as Araldite epoxy resin; 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 ultrathin 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 are 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 (cryofixed), 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 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. Such coatings include gold, gold/palladium, platinum, tungsten, graphite etc. and are especially important for the study of specimens with the scanning electron microscope. Another reason for coating, even when there is more than enough conductivity, is to improve contrast, a situation more common with the operation of a FESEM (field emission SEM).

Disadvantages

Pseudocolored SEM image of the feeding basket of Antarctic krill. Real electron microscope images do not carry any color information; they are greyscale. The first degree filter setae carry in v-form two rows of second degree setae, pointing towards the inside of the feeding basket. The purple ball is one micrometer in diameter. To display the total area of this structure one would have to tile this image 7500 times.

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. They are dynamic rather than static in their operation, requiring extremely stable high-voltage supplies, extremely stable currents to each electromagnetic coil/lens, continuously-pumped high- or ultra-high-vacuum systems, and a cooling water supply circulation through the lenses and pumps. As they are very sensitive to vibration and external magnetic fields, microscopes designed to achieve high resolutions must be housed in stable buildings (sometimes underground) with special services such as magnetic field cancelling systems. Some desktop low voltage electron microscopes have TEM capabilities at very low voltages (around 5 kV) without stringent voltage supply, lens coil current, cooling water or vibration isolation requirements and as such are much less expensive to buy and far easier to install and maintain, but do not have the same ultra-high (atomic scale) resolution capabilities as the larger instruments.

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), wet environment.

Scanning electron microscopes usually image conductive or semi-conductive materials best. Non-conductive materials can be imaged by an environmental scanning electron microscope. A common preparation technique is to coat the sample with a several-nanometer layer of conductive material, such as gold, from a sputtering machine; however, this process has the potential to disturb delicate samples.

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. In addition, higher-resolution work has been directly compared to results from X-ray crystallography, providing independent confirmation of the validity of this technique.[citation needed] Since the 1980s, analysis of cryofixed, vitrified specimens has also become increasingly used by scientists, further confirming the validity of this technique.[13][14][15]

Applications

Semiconductor and data storage

Biology and life sciences

Research

Industry

  • High-resolution imaging
  • 2D & 3D micro-characterization
  • Macro sample to nanometer metrology
  • Particle detection and characterization
  • Direct beam-writing fabrication
  • Dynamic materials experiments
  • Sample preparation
  • Forensics
  • Mining (mineral liberation analysis)
  • Chemical/Petrochemical

See also

References

  1. ^ a b Ernst Ruska Nobel Prize autobiography
  2. ^ Ernst Ruska (1986). "Ernst Ruska Autobiography". Nobel Foundation. http://nobelprize.org/nobel_prizes/physics/laureates/1986/ruska-autobio.html. Retrieved 2007-02-06. 
  3. ^ DH Kruger, P Schneck and HR Gelderblom (May 13, 2000). "Helmut Ruska and the visualisation of viruses". The Lancet 355 (9216): 1713–1717. doi:10.1016/S0140-6736(00)02250-9. 
  4. ^ M von Ardenne and D Beischer (1940). "Untersuchung von metalloxud-rauchen mit dem universal-elektronenmikroskop" (in German). Zeitschrift Electrochemie 46: 270–277. 
  5. ^ MIT biography of Hillier
  6. ^ Erni, Rolf (2009). "Atomic-Resolution Imaging with a Sub-50-pm Electron Probe". Physical Review Letters 102: 096101. doi:10.1103/PhysRevLett.102.096101. 
  7. ^ The Scale of Things, DOE Office of Basic Energy Sciences (BES).
  8. ^ Michael A. O'Keefe, Lawrence F. Allard. Sub-Ångstrom Electron Microscopy for Sub-Ångstrom Nano-Metrology. http://www.osti.gov/bridge/servlets/purl/821768-E3YVgN/native/821768.pdf. 
  9. ^ Scanning electron microscopy 1928 - 1965
  10. ^ NCEM National Center for Electron Microscopy: SPLEEM
  11. ^ Nebesářová1, Jana; Vancová, Marie (2007). "How to Observe Small Biological Objects in Low Voltage Electron Microscope". Microscopy and Microanalysis 13 (3): 248-249. doi:10.1017/S143192760708124X. 
  12. ^ Drummy, Lawrence, F.; Yang, Junyan; Martin, David C. (2004). "Low-voltage electron microscopy of polymer and organic molecular thin films". Ultramicroscopy 99: 247-256. doi:10.1016/j.ultramic.2004.01.011. 
  13. ^ Adrian, Marc; Dubochet, Jacques; Lepault, Jean; McDowall, Alasdair W. (1984). "Cryo-electron microscopy of viruses". Nature 308 (5954): 32–36. doi:10.1038/308032a0. 
  14. ^ Sabanay, I.; Arad, T.; Weiner, S.; Geiger, B. (1991). "Study of vitrified, unstained frozen tissue sections by cryoimmunoelectron microscopy". Journal of Cell Science 100 (1): 227–236. PMID 1795028. http://jcs.biologists.org/cgi/content/abstract/100/1/227. 
  15. ^ Kasas, S.; Dumas, G.; Dietler, G.; Catsicas, S.; Adrian, M. (2003). "Vitrification of cryoelectron microscopy specimens revealed by high-speed photographic imaging". Journal of Microscopy 211 (1): 48–53. doi:10.1046/j.1365-2818.2003.01193.x. 

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