Electron microscope

Electron microscope

Overview
An electron microscope is a type of microscope
Microscope
A microscope is an instrument used to see objects that are too small for the naked eye. The science of investigating small objects using such an instrument is called microscopy...

 that uses a beam of electron
Electron
The electron is a subatomic particle with a negative elementary electric charge. It has no known components or substructure; in other words, it is generally thought to be an elementary particle. An electron has a mass that is approximately 1/1836 that of the proton...

s to illuminate the specimen and produce a magnified image. Electron microscopes (EM) have a greater resolving power
Angular resolution
Angular resolution, or spatial resolution, describes the ability of any image-forming device such as an optical or radio telescope, a microscope, a camera, or an eye, to distinguish small details of an object...

 than a light-powered optical microscope
Optical microscope
The optical microscope, often referred to as the "light microscope", is a type of microscope which uses visible light and a system of lenses to magnify images of small samples. Optical microscopes are the oldest design of microscope and were possibly designed in their present compound form in the...

, because electrons have wavelengths about 100,000 times shorter than visible light (photon
Photon
In physics, a photon is an elementary particle, the quantum of the electromagnetic interaction and the basic unit of light and all other forms of electromagnetic radiation. It is also the force carrier for the electromagnetic force...

s), and can achieve better than 50 pm
Picometre
A picometre is a unit of length in the metric system, equal to one trillionth, i.e. of a metre, which is the current SI base unit of length...

 resolution and magnification
Magnification
Magnification is the process of enlarging something only in appearance, not in physical size. This enlargement is quantified by a calculated number also called "magnification"...

s of up to about 10,000,000x, whereas ordinary, non-confocal light microscopes are limited by diffraction
Diffraction
Diffraction refers to various phenomena which occur when a wave encounters an obstacle. Italian scientist Francesco Maria Grimaldi coined the word "diffraction" and was the first to record accurate observations of the phenomenon in 1665...

 to about 200 nm resolution and useful magnifications below 2000x.

The electron microscope uses electrostatic
Electrostatic lens
An electrostatic lens is a device that assists in the transport of charged particles. For instance, it can guide electrons emitted from a sample to an electron analyzer, analogous to the way an optical lens assists in the transport of light in an optical instrument. The recent development of...

 and electromagnetic
Electromagnetism
Electromagnetism is one of the four fundamental interactions in nature. The other three are the strong interaction, the weak interaction and gravitation...

 "lenses" to control the electron beam and focus it to form an image.
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Encyclopedia
An electron microscope is a type of microscope
Microscope
A microscope is an instrument used to see objects that are too small for the naked eye. The science of investigating small objects using such an instrument is called microscopy...

 that uses a beam of electron
Electron
The electron is a subatomic particle with a negative elementary electric charge. It has no known components or substructure; in other words, it is generally thought to be an elementary particle. An electron has a mass that is approximately 1/1836 that of the proton...

s to illuminate the specimen and produce a magnified image. Electron microscopes (EM) have a greater resolving power
Angular resolution
Angular resolution, or spatial resolution, describes the ability of any image-forming device such as an optical or radio telescope, a microscope, a camera, or an eye, to distinguish small details of an object...

 than a light-powered optical microscope
Optical microscope
The optical microscope, often referred to as the "light microscope", is a type of microscope which uses visible light and a system of lenses to magnify images of small samples. Optical microscopes are the oldest design of microscope and were possibly designed in their present compound form in the...

, because electrons have wavelengths about 100,000 times shorter than visible light (photon
Photon
In physics, a photon is an elementary particle, the quantum of the electromagnetic interaction and the basic unit of light and all other forms of electromagnetic radiation. It is also the force carrier for the electromagnetic force...

s), and can achieve better than 50 pm
Picometre
A picometre is a unit of length in the metric system, equal to one trillionth, i.e. of a metre, which is the current SI base unit of length...

 resolution and magnification
Magnification
Magnification is the process of enlarging something only in appearance, not in physical size. This enlargement is quantified by a calculated number also called "magnification"...

s of up to about 10,000,000x, whereas ordinary, non-confocal light microscopes are limited by diffraction
Diffraction
Diffraction refers to various phenomena which occur when a wave encounters an obstacle. Italian scientist Francesco Maria Grimaldi coined the word "diffraction" and was the first to record accurate observations of the phenomenon in 1665...

 to about 200 nm resolution and useful magnifications below 2000x.

The electron microscope uses electrostatic
Electrostatic lens
An electrostatic lens is a device that assists in the transport of charged particles. For instance, it can guide electrons emitted from a sample to an electron analyzer, analogous to the way an optical lens assists in the transport of light in an optical instrument. The recent development of...

 and electromagnetic
Electromagnetism
Electromagnetism is one of the four fundamental interactions in nature. The other three are the strong interaction, the weak interaction and gravitation...

 "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
Cell (biology)
The cell is the basic structural and functional unit of all known living organisms. It is the smallest unit of life that is classified as a living thing, and is often called the building block of life. The Alberts text discusses how the "cellular building blocks" move to shape developing embryos....

, large molecules, biopsy
Biopsy
A biopsy is a medical test involving sampling of cells or tissues for examination. It is the medical removal of tissue from a living subject to determine the presence or extent of a disease. The tissue is generally examined under a microscope by a pathologist, and can also be analyzed chemically...

 samples, metals, and crystals. Industrially, the electron microscope is often used for quality control and failure analysis.

History


The electron microscope was invented and patented by Hungarian physicist Leo Szilárd
Leó Szilárd
Leó Szilárd was an Austro-Hungarian physicist and inventor who conceived the nuclear chain reaction in 1933, patented the idea of a nuclear reactor with Enrico Fermi, and in late 1939 wrote the letter for Albert Einstein's signature that resulted in the Manhattan Project that built the atomic bomb...

 who declined to construct it. Instead, German physicist Ernst Ruska
Ernst Ruska
Ernst August Friedrich Ruska 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...

 and electrical engineer Max Knoll
Max Knoll
Max Knoll was a German electrical engineer.Knoll was born in Wiesbaden and studied in Munich and at the Technical University of Berlin, where he obtained his doctorate in the Institute for High Voltage Technology...

 constructed the prototype
Prototype
A prototype is an early sample or model built to test a concept or process or to act as a thing to be replicated or learned from.The word prototype derives from the Greek πρωτότυπον , "primitive form", neutral of πρωτότυπος , "original, primitive", from πρῶτος , "first" and τύπος ,...

 electron microscope in 1931, capable of four-hundred-power magnification; the apparatus was a practical application of the principles of electron microscopy. Two years later, in 1933, Ruska built an electron microscope that exceeded the resolution attainable with an optical (lens) microscope. Moreover, Reinhold Rudenberg
Reinhold Rudenberg
Reinhold Rudenberg was a German-American electrical engineer and inventor, credited with many innovations in the electric power and related fields...

, 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
Poliomyelitis
Poliomyelitis, often called polio or infantile paralysis, is an acute viral infectious disease spread from person to person, primarily via the fecal-oral route...

 virus.

In 1932, Ernst Lubcke of Siemens & Halske
Siemens & Halske
Siemens & Halske AG was a German electrical engineering company that later became part of Siemens AG.It was founded on 12 October 1847 as Telegraphen-Bauanstalt von Siemens & Halske by Ernst Werner von Siemens and Johann Georg Halske...

 built and obtained images from a prototype electron microscope, applying concepts described in the Rudenberg patent applications. Five years later (1937), the firm financed the work of Ernst Ruska and Bodo von Borries, and employed Helmut Ruska
Helmut Ruska
Helmut Ruska was a German physician and biologist from Heidelberg. After earning his medical degree, he spent several years working as a physician at hospitals in Heidelberg and Berlin...

 (Ernst’s brother) to develop applications for the microscope, especially with biologic specimens. Also in 1937, Manfred von Ardenne
Manfred von Ardenne
Manfred von Ardenne was a German research and applied physicist and inventor. He took out approximately 600 patents in fields including electron microscopy, medical technology, nuclear technology, plasma physics, and radio and television technology...

 pioneered the scanning electron microscope
Scanning electron microscope
A scanning electron microscope is a type of electron microscope that images a sample by scanning it with a high-energy beam of electrons in a raster scan pattern...

. The first practical electron microscope was constructed in 1938, at the University of Toronto
University of Toronto
The University of Toronto is a public research university in Toronto, Ontario, Canada, situated on the grounds that surround Queen's Park. It was founded by royal charter in 1827 as King's College, the first institution of higher learning in Upper Canada...

, by Eli Franklin Burton
Eli Franklin Burton
Eli Franklin Burton, was a Canadian physicist.Burton was born in Green River, township of Pickering, Ontario, Canada. He graduated from the University of Toronto in 1901. From 1904 to 1906 he studied colloids with J. J. Thomson at the Cavendish Laboratory at the University of Cambridge, writing...

 and students Cecil Hall, James Hillier
James Hillier
James Hillier, was a Canadian-born scientist and inventor who designed and built, with Albert Prebus, the first successful high-resolution electron microscope in North America in 1938....

, and Albert Prebus; and Siemens produced the first commercial transmission electron microscope (TEM) in 1939. Although contemporary electron microscopes are capable of two million-power magnification, as scientific instruments, they remain based upon Ruska’s prototype
Prototype
A prototype is an early sample or model built to test a concept or process or to act as a thing to be replicated or learned from.The word prototype derives from the Greek πρωτότυπον , "primitive form", neutral of πρωτότυπος , "original, primitive", from πρῶτος , "first" and τύπος ,...

.

Transmission electron microscope (TEM)



The original form of electron microscope, the transmission electron microscope (TEM) uses a high voltage
High voltage
The term high voltage characterizes electrical circuits in which the voltage used is the cause of particular safety concerns and insulation requirements...

 electron beam to create an image. The electrons are emitted by an electron gun
Electron gun
An electron gun is an electrical component that produces an electron beam that has a precise kinetic energy and is most often used in television sets and computer displays which use cathode ray tube technology, as well as in other instruments, such as electron microscopes and particle...

, commonly fitted with a tungsten
Tungsten
Tungsten , also known as wolfram , is a chemical element with the chemical symbol W and atomic number 74.A hard, rare metal under standard conditions when uncombined, tungsten is found naturally on Earth only in chemical compounds. It was identified as a new element in 1781, and first isolated as...

 filament cathode
Cathode
A cathode is an electrode through which electric current flows out of a polarized electrical device. Mnemonic: CCD .Cathode polarity is not always negative...

 as the electron source. The electron beam is accelerated by an anode
Anode
An anode is an electrode through which electric current flows into a polarized electrical device. Mnemonic: ACID ....

 typically at +100 keV
Electronvolt
In physics, the electron volt is a unit of energy equal to approximately joule . By definition, it is equal to the amount of kinetic energy gained by a single unbound electron when it accelerates through an electric potential difference of one volt...

 (40 to 400 keV) with respect to the cathode, focused by electrostatic and electromagnetic
Electromagnetism
Electromagnetism is one of the four fundamental interactions in nature. The other three are the strong interaction, the weak interaction and gravitation...

 lenses, and transmitted through the specimen that is in part transparent to electrons and in part scatters
Electron scattering
Electron scattering is the process whereby an electron is deflected from its original trajectory. As they are charged particles, they are subject to electromagnetic forces.-Phenomena:...

 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
Phosphor
A phosphor, most generally, is a substance that exhibits the phenomenon of luminescence. Somewhat confusingly, this includes both phosphorescent materials, which show a slow decay in brightness , and fluorescent materials, where the emission decay takes place over tens of nanoseconds...

 or scintillator
Scintillator
A scintillator is a special material, which exhibits scintillation—the property of luminescence when excited by ionizing radiation. Luminescent materials, when struck by an incoming particle, absorb its energy and scintillate, i.e., reemit the absorbed energy in the form of light...

 material such as zinc sulfide. Alternatively, the image can be photographically recorded by exposing a photographic film
Photographic film
Photographic film is a sheet of plastic coated with an emulsion containing light-sensitive silver halide salts with variable crystal sizes that determine the sensitivity, contrast and resolution of the film...

 or plate
Photographic plate
Photographic plates preceded photographic film as a means of photography. A light-sensitive emulsion of silver salts was applied to a glass plate. This form of photographic material largely faded from the consumer market in the early years of the 20th century, as more convenient and less fragile...

 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
Charge-coupled device
A charge-coupled device is a device for the movement of electrical charge, usually from within the device to an area where the charge can be manipulated, for example conversion into a digital value. This is achieved by "shifting" the signals between stages within the device one at a time...

) 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
Spherical aberration
thumb|right|Spherical aberration. A perfect lens focuses all incoming rays to a point on the [[Optical axis|optic axis]]. A real lens with spherical surfaces suffers from spherical aberration: it focuses rays more tightly if they enter it far from the optic axis than if they enter closer to the...

, 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
Ångström
The angstrom or ångström, is a unit of length equal to 1/10,000,000,000 of a meter . Its symbol is the Swedish letter Å....

 (50 picometre
Picometre
A picometre is a unit of length in the metric system, equal to one trillionth, i.e. of a metre, which is the current SI base unit of length...

s) and magnifications above 50 million times. The ability to determine the positions of atoms within materials has made the HRTEM an important tool for nano-technologies research and development.

An important mode of TEM utilization is electron diffraction
Electron diffraction
Electron diffraction refers to the wave nature of electrons. However, from a technical or practical point of view, it may be regarded as a technique used to study matter by firing electrons at a sample and observing the resulting interference pattern...

. 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



Unlike the TEM, where electrons of the high voltage beam carry the image of the specimen, the electron beam of the scanning electron microscope
Scanning electron microscope
A scanning electron microscope is a type of electron microscope that images a sample by scanning it with a high-energy beam of electrons in a raster scan pattern...

 (SEM) 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 scan
Raster scan
A raster scan, or raster scanning, is the rectangular pattern of image capture and reconstruction in television. By analogy, the term is used for raster graphics, the pattern of image storage and transmission used in most computer bitmap image systems...

ning). 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
Secondary emission
Secondary emission in physics is a phenomenon where primary incident particles of sufficient energy, when hitting a surface or passing through some material, induce the emission of secondary particles. The primary particles are often charged particles like electrons or ions. If the secondary...

 and high-energy backscattered electrons, light emission (cathodoluminescence
Cathodoluminescence
Cathodoluminescence is an optical and electrical phenomenon whereby a beam of electrons is generated by an electron gun and then impacts on a luminescent material such as a phosphor, causing the material to emit visible light. The most common example is the screen of a television...

) or X-ray
X-ray
X-radiation is a form of electromagnetic radiation. X-rays have a wavelength in the range of 0.01 to 10 nanometers, corresponding to frequencies in the range 30 petahertz to 30 exahertz and energies in the range 120 eV to 120 keV. They are shorter in wavelength than UV rays and longer than gamma...

 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


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
Elastic scattering
In scattering theory and in particular in particle physics, elastic scattering is one of the specific forms of scattering. In this process, the kinetic energy of the incident particles is conserved, only their direction of propagation is modified .-Electron elastic scattering:When an alpha particle...

 is detected. This technique is typically coupled with reflection high energy electron diffraction
RHEED
Reflection high-energy electron diffraction is a technique used to characterize the surface of crystalline materials. RHEED systems gather information only from the surface layer of the sample, which distinguishes RHEED from other materials characterization methods that also rely on diffraction of...

 (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.

Scanning transmission electron microscope



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
Annular dark-field imaging
Annular dark-field imaging is a method of mapping samples in a scanning transmission electron microscope . These images are formed by collecting scattered electrons with an annular dark-field detector....

, and other analytical techniques, but also means that image data is acquired in serial rather than in parallel fashion.

Low-voltage electron microscope


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.

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:
  • Chemical fixation
    Fixation (histology)
    In the fields of histology, pathology, and cell biology, fixation is a chemical process by which biological tissues are preserved from decay, thereby preventing autolysis or putrefaction...

    – for biological specimens aims to stabilize the specimen's mobile macromolecular structure by chemical crosslinking of protein
    Protein
    Proteins are biochemical compounds consisting of one or more polypeptides typically folded into a globular or fibrous form, facilitating a biological function. A polypeptide is a single linear polymer chain of amino acids bonded together by peptide bonds between the carboxyl and amino groups of...

    s with aldehyde
    Aldehyde
    An aldehyde is an organic compound containing a formyl group. This functional group, with the structure R-CHO, consists of a carbonyl center bonded to hydrogen and an R group....

    s such as formaldehyde
    Formaldehyde
    Formaldehyde is an organic compound with the formula CH2O. It is the simplest aldehyde, hence its systematic name methanal.Formaldehyde is a colorless gas with a characteristic pungent odor. It is an important precursor to many other chemical compounds, especially for polymers...

     and glutaraldehyde
    Glutaraldehyde
    Glutaraldehyde is an organic compound with the formula CH22. A pungent colorless oily liquid, glutaraldehyde is used to disinfect medical and dental equipment...

    , and lipid
    Lipid
    Lipids constitute a broad group of naturally occurring molecules that include fats, waxes, sterols, fat-soluble vitamins , monoglycerides, diglycerides, triglycerides, phospholipids, and others...

    s with osmium tetroxide.
  • Negative stain
    Negative stain
    Negative staining is an established method, often used in diagnostic microscopy, for contrasting a thin specimen with an optically opaque fluid....

    – suspensions containing 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
    Cryofixation
    Cryofixation is a technique for fixation or stabilisation of biological materials as the first step in specimen preparation for electron microscopy...

    – freezing a specimen so rapidly, to liquid nitrogen
    Liquid nitrogen
    Liquid nitrogen is nitrogen in a liquid state at a very low temperature. It is produced industrially by fractional distillation of liquid air. Liquid nitrogen is a colourless clear liquid with density of 0.807 g/mL at its boiling point and a dielectric constant of 1.4...

     or even liquid helium
    Liquid helium
    Helium exists in liquid form only at extremely low temperatures. The boiling point and critical point depend on the isotope of the helium; see the table below for values. The density of liquid helium-4 at its boiling point and 1 atmosphere is approximately 0.125 g/mL Helium-4 was first liquefied...

     temperatures, that the water forms vitreous (non-crystalline) ice
    Amorphous ice
    Amorphous ice is an amorphous solid form of water, meaning it consists of water molecules that are randomly arranged like the atoms of common glass. Everyday ice is a crystalline material where the atoms are regularly arranged in a lattice whereas amorphous ice is distinguished by a lack of...

    . This preserves the specimen in a snapshot of its solution state. An entire field called cryo-electron microscopy
    Cryo-electron microscopy
    Cryo-electron microscopy , or electron cryomicroscopy, is a form of transmission electron microscopy where the sample is studied at cryogenic temperatures...

     has branched from this technique. With the development of cryo-electron microscopy of vitreous sections
    Cryo-electron microscopy
    Cryo-electron microscopy , or electron cryomicroscopy, is a form of transmission electron microscopy where the sample is studied at cryogenic temperatures...

     (CEMOVIS), it is now possible to observe samples from virtually any biological specimen close to its native state.
  • Dehydrationfreeze drying
    Freeze drying
    Freeze-drying is a dehydration process typically used to preserve a perishable material or make the material more convenient for transport...

    , or replacement of water
    Water
    Water is a chemical substance with the chemical formula H2O. A water molecule contains one oxygen and two hydrogen atoms connected by covalent bonds. Water is a liquid at ambient conditions, but it often co-exists on Earth with its solid state, ice, and gaseous state . Water also exists in a...

     with organic solvents such as ethanol
    Ethanol
    Ethanol, also called ethyl alcohol, pure alcohol, grain alcohol, or drinking alcohol, is a volatile, flammable, colorless liquid. It is a psychoactive drug and one of the oldest recreational drugs. Best known as the type of alcohol found in alcoholic beverages, it is also used in thermometers, as a...

     or acetone
    Acetone
    Acetone is the organic compound with the formula 2CO, a colorless, mobile, flammable liquid, the simplest example of the ketones.Acetone is miscible with water and serves as an important solvent in its own right, typically as the solvent of choice for cleaning purposes in the laboratory...

    , 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
    Resin
    Resin in the most specific use of the term is a hydrocarbon secretion of many plants, particularly coniferous trees. Resins are valued for their chemical properties and associated uses, such as the production of varnishes, adhesives, and food glazing agents; as an important source of raw materials...

     such as Araldite
    Araldite
    Araldite is a registered trademark of Huntsman Advanced Materials referring to their range of engineering and structural epoxy, acrylic, and polyurethane adhesives. The name was first used in 1946 for a two-part epoxy adhesive....

     epoxy
    Epoxy
    Epoxy, also known as polyepoxide, is a thermosetting polymer formed from reaction of an epoxide "resin" with polyamine "hardener". Epoxy has a wide range of applications, including fiber-reinforced plastic materials and general purpose adhesives....

     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
    Diamond
    In mineralogy, diamond is an allotrope of carbon, where the carbon atoms are arranged in a variation of the face-centered cubic crystal structure called a diamond lattice. Diamond is less stable than graphite, but the conversion rate from diamond to graphite is negligible at ambient conditions...

     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
    Lead
    Lead is a main-group element in the carbon group with the symbol Pb and atomic number 82. Lead is a soft, malleable poor metal. It is also counted as one of the heavy metals. Metallic lead has a bluish-white color after being freshly cut, but it soon tarnishes to a dull grayish color when exposed...

    , uranium
    Uranium
    Uranium is a silvery-white metallic chemical element in the actinide series of the periodic table, with atomic number 92. It is assigned the chemical symbol U. A uranium atom has 92 protons and 92 electrons, of which 6 are valence electrons...

     or tungsten
    Tungsten
    Tungsten , also known as wolfram , is a chemical element with the chemical symbol W and atomic number 74.A hard, rare metal under standard conditions when uncombined, tungsten is found naturally on Earth only in chemical compounds. It was identified as a new element in 1781, and first isolated as...

     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
    Hypochlorite
    The hypochlorite ion, also known as chlorate anion is ClO−. A hypochlorite compound is a chemical compound containing this group, with chlorine in oxidation state +1.Hypochlorites are the salts of hypochlorous acid...

     solution or SDS
    Sodium dodecyl sulfate
    Sodium dodecyl sulfate , sodium laurilsulfate or sodium lauryl sulfate is an organic compound with the formula CH311OSO3Na). It is an anionic surfactant used in many cleaning and hygiene products...

     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
    Argon
    Argon is a chemical element represented by the symbol Ar. Argon has atomic number 18 and is the third element in group 18 of the periodic table . Argon is the third most common gas in the Earth's atmosphere, at 0.93%, making it more common than carbon dioxide...

    ) at the surface from an angle and sputtering material from the surface. A subclass of this is focused ion beam
    Focused ion beam
    Focused ion beam, also known as FIB, is a technique used particularly in the semiconductor industry, materials science and increasingly in the biological field for site-specific analysis, deposition, and ablation of materials. An FIB setup is a scientific instrument that resembles a scanning...

     milling, where gallium
    Gallium
    Gallium is a chemical element that has the symbol Ga and atomic number 31. Elemental gallium does not occur in nature, but as the gallium salt in trace amounts in bauxite and zinc ores. A soft silvery metallic poor metal, elemental gallium is a brittle solid at low temperatures. As it liquefies...

     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. Coating is especially important for the study of specimens with the scanning electron microscope where electrons are accelerated by a relatively low voltage and therefore are affected more by the sample charging. Another reason for coating, even when there is more than enough conductivity, is to improve contrast, a situation more common with the operation of an FESEM (field emission SEM).

Disadvantages


Electron microscopes are expensive to build and maintain, but the capital and running costs of confocal light microscope
Confocal microscopy
Confocal microscopy is an optical imaging technique used to increase optical resolution and contrast of a micrograph by using point illumination and a spatial pinhole to eliminate out-of-focus light in specimens that are thicker than the focal plane. It enables the reconstruction of...

 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 relatively 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
Vacuum
In everyday usage, vacuum is a volume of space that is essentially empty of matter, such that its gaseous pressure is much less than atmospheric pressure. The word comes from the Latin term for "empty". A perfect vacuum would be one with no particles in it at all, which is impossible to achieve in...

, 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 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 an variable pressure (or environmental) scanning electron microscope.

Small, stable specimens such as carbon nanotubes, diatom
Diatom
Diatoms are a major group of algae, and are one of the most common types of phytoplankton. Most diatoms are unicellular, although they can exist as colonies in the shape of filaments or ribbons , fans , zigzags , or stellate colonies . Diatoms are producers within the food chain...

 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
Cryofixation
Cryofixation is a technique for fixation or stabilisation of biological materials as the first step in specimen preparation for electron microscopy...

, vitrified specimens has also become increasingly used by scientists, further confirming the validity of this technique.

Applications


Semiconductor and data storage
  • Circuit edit
  • Defect analysis
  • Failure analysis
    Failure analysis
    Failure analysis is the process of collecting and analyzing data to determine the cause of a failure. It is an important discipline in many branches of manufacturing industry, such as the electronics industry, where it is a vital tool used in the development of new products and for the improvement...


Biology and life sciences
  • Diagnostic electron microscopy
    Diagnostic electron microscopy
    The transmission electron microscope is used as an important diagnostic tool to screen human tissues at high magnification , often in conjunction with other methods, particularly light microscopy and immunofluorescence techniques...

  • Cryobiology
    Cryobiology
    Cryobiology is the branch of biology that studies the effects of low temperatures on living things. The word cryobiology is derived from the Greek words "cryo" = cold, "bios" = life, and "logos" = science. In practice, cryobiology is the study of biological material or systems at temperatures below...

  • Protein localization
  • Electron tomography
    Electron tomography
    Electron Tomography is a tomography technique for obtaining detailed 3D structures of subcellular macromolecular objects. Electron tomography is an extension of traditional transmission electron microscopy and uses a transmission electron microscope to collect the data...

  • Cellular tomography
  • Cryo-electron microscopy
    Cryo-electron microscopy
    Cryo-electron microscopy , or electron cryomicroscopy, is a form of transmission electron microscopy where the sample is studied at cryogenic temperatures...

  • Toxicology
    Toxicology
    Toxicology is a branch of biology, chemistry, and medicine concerned with the study of the adverse effects of chemicals on living organisms...

  • Biological production and viral load
    Viral load
    Viral load is a measure of the severity of a viral infection, and can be calculated by estimating the amount of virus in an involved body fluid. For example, it can be given in RNA copies per milliliter of blood plasma...

     monitoring
  • Particle analysis
  • Pharmaceutical QC
  • Structural biology
    Structural biology
    Structural biology is a branch of molecular biology, biochemistry, and biophysics concerned with the molecular structure of biological macromolecules, especially proteins and nucleic acids, how they acquire the structures they have, and how alterations in their structures affect their function...

  • 3D tissue imaging
  • Virology
    Virology
    Virology is the study of viruses and virus-like agents: their structure, classification and evolution, their ways to infect and exploit cells for virus reproduction, the diseases they cause, the techniques to isolate and culture them, and their use in research and therapy...

  • Vitrification
    Vitrification
    Vitrification is the transformation of a substance into a glass. Usually, it is achieved by rapidly cooling a liquid through the glass transition. Certain chemical reactions also result in glasses...



Research
  • Electron beam-induced deposition
  • Materials qualification
  • Materials and sample preparation
  • Nanoprototyping
  • Nanometrology
    Nanometrology
    Nanometrology is a subfield of metrology, concerned with the science of measurement at the nanoscale level. Nanometrology has a crucial role in order to produce nanomaterials and devices with a high degree of accuracy and reliability in nanomanufacturing....

  • Device testing and characterization

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
    Forensics
    Forensic science is the application of a broad spectrum of sciences to answer questions of interest to a legal system. This may be in relation to a crime or a civil action...

  • Mining
    Mining
    Mining is the extraction of valuable minerals or other geological materials from the earth, from an ore body, vein or seam. The term also includes the removal of soil. Materials recovered by mining include base metals, precious metals, iron, uranium, coal, diamonds, limestone, oil shale, rock...

     (mineral liberation analysis)
  • Chemical/Petrochemical
    Petrochemical
    Petrochemicals are chemical products derived from petroleum. Some chemical compounds made from petroleum are also obtained from other fossil fuels, such as coal or natural gas, or renewable sources such as corn or sugar cane....



See also


  • :Category:Electron microscope images
  • Electron energy loss spectroscopy
    Electron energy loss spectroscopy
    In electron energy loss spectroscopy a material is exposed to a beam of electrons with a known, narrow range of kinetic energies. Some of the electrons will undergo inelastic scattering, which means that they lose energy and have their paths slightly and randomly deflected...

     (EELS)
  • Energy filtered transmission electron microscopy
    Energy filtered transmission electron microscopy
    Energy-filtered transmission electron microscopy is a technique used in Transmission electron microscopy, in which only electrons of particular kinetic energies are used to form the image or diffraction pattern...

     (EFTEM)
  • Field emission microscope
  • HiRISE
    HiRISE
    High Resolution Imaging Science Experiment is a camera on board the Mars Reconnaissance Orbiter. The 65 kg , $40 million instrument was built under the direction of the University of Arizona's Lunar and Planetary Laboratory by Ball Aerospace & Technologies Corp....

  • Scanning tunneling microscope
    Scanning tunneling microscope
    A scanning tunneling microscope is an instrument for imaging surfaces at the atomic level. Its development in 1981 earned its inventors, Gerd Binnig and Heinrich Rohrer , the Nobel Prize in Physics in 1986. For an STM, good resolution is considered to be 0.1 nm lateral resolution and...

  • Scanning confocal electron microscopy
    Scanning confocal electron microscopy
    Scanning confocal electron microscopy is an electron microscopy technique analogous to scanning confocal optical microscopy . In this technique, the studied sample is illuminated by a focussed electron beam, as in other scanning microscopy techniques, such as scanning transmission electron...

  • Scanning electron microscope
    Scanning electron microscope
    A scanning electron microscope is a type of electron microscope that images a sample by scanning it with a high-energy beam of electrons in a raster scan pattern...

     (SEM)
  • Transmission Electron Aberration-corrected Microscope
    Transmission Electron Aberration-corrected Microscope
    Transmission Electron Aberration-Corrected Microscope is a collaborative research project between four US laboratories and two companies. It is based at the Lawrence Berkeley National Laboratory in Berkeley, California and involves Argonne National Laboratory, Oak Ridge National Laboratory and...

  • Electron diffraction
    Electron diffraction
    Electron diffraction refers to the wave nature of electrons. However, from a technical or practical point of view, it may be regarded as a technique used to study matter by firing electrons at a sample and observing the resulting interference pattern...

  • X-ray diffraction
  • X-ray microscope
    X-ray microscope
    An X-ray microscope uses electromagnetic radiation in the soft X-ray band to produce images of very small objects.Unlike visible light, X-rays do not reflect or refract easily, and they are invisible to the human eye. Therefore the basic process of an X-ray microscope is to expose film or use a...

  • X-ray photoelectron spectroscopy
    X-ray photoelectron spectroscopy
    X-ray photoelectron spectroscopy is a quantitative spectroscopic technique that measures the elemental composition, empirical formula, chemical state and electronic state of the elements that exist within a material...

     (XPS)
  • Microscope image processing
    Microscope image processing
    Microscope image processing is a broad term that covers the use of digital image processing techniques to process, analyze and present images obtained from a microscope. Such processing is now commonplace in a number of diverse fields such as medicine, biological research, cancer research, drug...

  • Microscopy
    Microscopy
    Microscopy is the technical field of using microscopes to view samples and objects that cannot be seen with the unaided eye...

  • Acronyms in microscopy
  • Nanoscience
  • Nanotechnology
    Nanotechnology
    Nanotechnology is the study of manipulating matter on an atomic and molecular scale. Generally, nanotechnology deals with developing materials, devices, or other structures possessing at least one dimension sized from 1 to 100 nanometres...

  • Surface science
    Surface science
    Surface science is the study of physical and chemical phenomena that occur at the interface of two phases, including solid–liquid interfaces, solid–gas interfaces, solid–vacuum interfaces, and liquid-gas interfaces. It includes the fields of surface chemistry and surface physics. Some related...

  • Ultramicroscopy
    Ultramicroscopy
    Ultramicroscopy is a peer-reviewed scientific journal in the field of electron microscopy....

    (journal)


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