Scanning electron microscope
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The scanning electron microscope (SEM) is a type of electron microscope capable of producing high-resolution images of a sample surface. Due to the manner in which the image is created, SEM images have a characteristic three-dimensional appearance and are useful for judging the surface structure of the sample.
History
The SEM was pioneered by Manfred von Ardenne in the 1930s [1] [1]. The instrument was further developed by Charles Oatley and first commercialized by Cambridge Instruments.
Scanning process
In a typical SEM, electrons are thermionically emitted from a tungsten or lanthanum hexaboride (LaB6) cathode and are accelerated towards an anode; alternatively, electrons can be emitted via field emission (FE). Tungsten is used because it has the highest melting point and lowest vapour pressure of all metals, thereby allowing it to be heated for electron emission. The electron beam, which typically has an energy ranging from a few hundred eV to 100 keV, is focused by one or two condenser lenses into a beam with a very fine focal spot sized 0.4 nm to 5 nm. The beam passes through pairs of scanning coils or pairs of deflector plates in the electron optical column, typically in the objective lens, which deflect the beam horizontally and vertically so that it scans in a raster fashion over a rectangular area of the sample surface. When the primary electron beam interacts with the sample, the electrons lose energy by repeated scattering and absorption within a teardrop-shaped volume of the specimen known as the interaction volume, which extends from less than 100 nm to around 5 µm into the surface. The size of the interaction volume depends on the electrons' landing energy, the atomic number of the specimen and the specimen's density. The energy exchange between the electron beam and the sample results in the emission of electrons and electromagnetic radiation, which can be detected to produce an image, as described below.
Sample preparation
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:
- 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 virtually any biological specimen close to its native state.
- Dehydration – replacing water with organic solvents such as ethanol or acetone.
- Embedding – infiltration of the tissue with a resin such as araldite or epoxy for sectioning. After this embedding process begins, the specimen must be polished to a mirror-like finish using ultra-fine abrasives. The polishing process must be done accordingly, or it may lead to scratches imposing on the image quality.
- 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 usually stained "en bloc" before embedding and also later stained directly after sectioning by brief exposure to aqueous (or alcoholic) solutions of the heavy metal stains.
- 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 EM grids, dried then viewed in the TEM.
- Ion Beam Milling – A Focused ion beam (such as gallium ions) are used to sputter material from a specific region of the sample, for example through a device within a microprocessor, to expose an underlying feature to view. 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). When an osmium coater is used, a layer far thinner than would be possible with any of the previously mentioned sputtered coatings is possible.[1]
Due to the loss of information, gold coating has until recently been considered a semi-destructive process, as it has not been possible to remove the coating without intensive handling, often damaging the specimen; a simple but novel technique involving potassium cyanide has recently been reported to permit such removal.[1]
Alternative techniques, for example the low-vacuum environmental SEM, allow samples to be imaged without such plating and without the loss of natural contrast arising from the beam-specimen interaction. Gold has a high atomic number and produces high topographic contrast and resolution but the information thus produced can obscure the underlying fine detail of the specimen under examination.
Detection of secondary electrons
The most common imaging mode monitors low energy (<50 eV) secondary electrons. Due to their low energy, these electrons originate within a few nanometers from the surface. The electrons are detected by an Everhart-Thornley detector which is a type of scintillator-photomultiplier device and the resulting signal is rendered into a two-dimensional intensity distribution that can be viewed and saved as a Digital image. This process relies on a raster-scanned primary beam. The brightness of the signal depends on the number of secondary electrons reaching the detector. If the beam enters the sample perpendicular to the surface, then the activated region is uniform about the axis of the beam and a certain number of electrons "escape" from within the sample. As the angle of incidence increases, the "escape" distance of one side of the beam will decrease, and more secondary electrons will be emitted. Thus steep surfaces and edges tend to be brighter than flat surfaces, which results in images with a well-defined, three-dimensional appearance. Using this technique, resolutions less than 1 nm are possible.
Detection of backscattered electrons
Backscattered electrons consist of high-energy electrons originating in the electron beam, that are reflected or back-scattered out of the specimen interaction volume. Backscattered electrons may be used to detect contrast between areas with different chemical compositions, especially when the average atomic number of the various regions is different, since the brightness of the BSE image tends to increase with the atomic number.
Backscattered electrons can also be used to form an electron backscatter diffraction (EBSD) image. This image can be used to determine the crystallographic structure of the specimen.
There are fewer backscattered electrons emitted from a sample than secondary electrons. The number of backscattered electrons leaving the sample surface upward might be significantly lower than those that follow trajectories toward the sides. Additionally, in contrast with the case with secondary electrons, the collection efficiency of backscattered electrons cannot be significantly improved by a positive bias common on Everhart-Thornley detectors. This detector positioned on one side of the sample has low collection efficiency for backscattered electrons due to small acceptance angles. The use of a dedicated backscattered electron detector above the sample in a "doughnut" type arrangement, with the electron beam passing through the hole of the doughnut, greatly increases the solid angle of collection and allows for the detection of more backscattered electrons.
Beam-injection analysis of semiconductors
The nature of the SEM's probe, energetic electrons, makes it uniquely suited to examining the optical and electronic properties of semiconductor materials. The high-energy electrons from the SEM beam will inject charge carriers into the semiconductor. Thus, beam electrons lose energy by promoting electrons from the valence band into the conduction band, leaving behind holes.
In a direct bandgap material, recombination of these electron-hole pairs will result in cathodoluminescence; if the sample contains an internal electric field, such as is present at a p-n junction, the SEM beam injection of carriers will cause electron beam induced current (EBIC) to flow.
Cathodoluminescence and EBIC are referred to as "beam-injection" techniques, and are very powerful probes of the optoelectronic behavior of semiconductors, particularly for studying nanoscale features and defects.
Cathodoluminescence
Cathodoluminescence, the emission of light when atoms excited by high-energy electrons return to their ground state, is analogous to UV-induced fluorescence, and some materials such as zinc sulphide and some fluorescent dyes, exhibit both phenomena. Cathodoluminescence is most commonly experienced in everyday life as the light emission from the inner surface of the cathode ray tube in television sets and computer CRT monitors. In the SEM, CL detectors either collect all light emitted by the specimen, or can analyse the wavelengths emitted by the specimen and display a spectrum or an image of the cathodoluminescence in real colour.
X-ray microanalysis
X-rays, which are also produced by the interaction of electrons with the sample, may also be detected in an SEM equipped for energy-dispersive X-ray spectroscopy or wavelength dispersive X-ray spectroscopy.
Resolution of the SEM
The spatial resolution of the SEM depends on the size of the electron spot, which in turn depends on the magnetic electron-optical system which produces the scanning beam. The resolution is also limited by the size of the interaction volume, or the extent to which the material interacts with the electron beam. The spot size and the interaction volume both might be large compared to the distances between atoms, so the resolution of the SEM is not high enough to image individual atoms, as is possible in the transmission electron microscope (TEM). The SEM has compensating advantages, though, including the ability to image a comparatively large area of the specimen; the ability to image bulk materials (not just thin films or foils); and the variety of analytical modes available for measuring the composition and nature of the specimen. Depending on the instrument, the resolution can fall somewhere between less than 1 nm and 20 nm. In general, SEM images are easier to interpret than TEM images.
Environmental SEM
Conventional SEM requires samples to be imaged under vacuum, which means that samples that would produce a significant amount of vapour, e.g. biological samples, need to be either dried or cryogenically frozen. This means that processes involving transitions to or from liquids or gases, such as the drying of adhesives or melting of alloys, liquid transport, chemical reactions and solid-air-gas systems in general could not be observed.
The first commercial development of the Environmental SEM (ESEM) in the late 1980s [1] [1] allowed samples to be observed in low-pressure gaseous environments (e.g. 1-50 Torr) and high relative humidity (up to 100%). This was made possible by the development of a secondary-electron detector [1] [1] capable of operating in the presence of water vapour and by the use of pressure-limiting apertures with differential pumping in the path of the electron beam to separate the vacuum regions around the gun and lenses from the sample chamber.
The first commercial ESEMs were produced by the ElectroScan Corporation in USA in 1988 [1]. ElectroScan were later taken over by Philips (now FEI Company) in 1996 [1].
ESEM is especially useful for non-metallic and biological materials because coating with carbon or gold is unnecessary. Plastics and Elastomers can now be routinely examined, as can biological samples. Coating is irreversible, and may reduce the value of the results obtained. Thus very small details on the surface of the sample may be concealed by the coating, let alone that coating is done under vacuum, which drastically alters hydrated specimens.
See also
References
External links
General
- Notes on the SEM Notes covering all aspects of the SEM
History
- Microscopy History links from the University of Alabama Department of Biological Sciences
- The Early History and Development of SEM from Cambridge University Engineering Department
- Milestones in the History of Electron Microscopy from the Swiss Federal Institute of Zürich
- Environmental Scanning Electron Microscope (ESEM) history
Images
- Tescan Image Gallery Some great SEM images of various specimens, as well as analytical results
- Dennis Kunkel Microscopy, Inc. Large collection of SEM images - mostly false colour
- Jeol SEM Images Twelve SEM images of various specimens
- SEM Lab at the Smithsonian National Museum of Natural History; includes a gallery of images
- Rippel Electron Microscope Facility Many dozens of (mostly biological) SEM images from Dartmouth College.
Gallery of SEM images
The following are examples of images taken using a scanning electron microscope.
Diatom0D.JPG
SEM Picture of a Diatom at magnification of 5000X. |
 False coloured SEM image of soybean cyst nematode and egg. |
||
 SEM image of an ant head. |
Anthophyllite asbestos SEM.jpg
SEM image of asbestos fibers. |
Krilleyekils.jpg
Compound eye of Antarctic krill Euphausia superba. |
Krillommatkils.jpg
Ommatidia of Antarctic krill eye. |
Nematocyst-discharged.png
SEM image of a discharged nematocyst. |
SEMFF.jpg
SEM image of the upper body of a Drosophila. |
FFEYE2.jpg
SEM images of the compound eye of a Drosophila. |
Spiraclevalves.jpg
SEM image of a cricket's spiracle valve. |
 SEM image of normal circulating human blood. |
HederelloidSEM.jpg
SEM image of a hederelloid from the Devonian of Michigan (largest tube diameter is 0.75 mm). |
BSEGlassInclusionSb.jpg
Backscattered Electron image of an Antimony rich region in a fragment of ancient glass. |
SEGlassCorrosion.jpg
SEM image of the corrosion layer on the surface of an ancient glass fragment, note the laminar structure of the corrosion layer. |
Photoresist SEM micrograph.JPG
SEM image of a photoresist layer used in semiconductor manufacturing |
de:Rasterelektronenmikroskopfa:میکروسکوپ الکترونی روبشی fr:Microscopie électronique à balayage it:Microscopio elettronico a scansione nl:Rasterelektronenmicroscoop ja:走査型電子顕微鏡sl:Vrstični elektronski mikroskop vi:Kính hiển vi điện tử quét uk:Скануючий електронний мікроскоп
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Some of the initial content on this page may be incorporated in part from copyleft sources in the public domain including wikis such as Wikipedia and AskDrWiki. Drug information for patients came from the The National Library of Medicine. Infectious disease information may have come from the Centers for Disease Control (CDC). Differential Diagnoses are drawn from clinicians as well as an amalgamation of 3 sources: 1.The Disease Database; 2. Kahan, Scott, Smith, Ellen G. In A Page: Signs and Symptoms. Malden, Massachusetts: Blackwell Publishing, 2004:3; 3. Sailer, Christian, Wasner, Susanne. Differential Diagnosis Pocket. Hermosa Beach, CA: Borm Bruckmeir Publishing LLC, 2002:7 .
