![Ernst Ruska Electron Microscope - Deutsches Museum - Munich-edit](https://upload.wikimedia.org/wikipedia/commons/1/1f/Ernst_Ruska_Electron_Microscope_-_Deutsches_Museum_-_Munich-edit.jpg)
An electron microscope is a
microscope
A microscope () is a laboratory instrument used to examine objects that are too small to be seen by the naked eye. Microscopy is the science of investigating small objects and structures using a microscope. Microscopic means being invisi ...
that uses a beam of accelerated
electron
The electron ( or ) is a subatomic particle with a negative one elementary electric charge. Electrons belong to the first generation of the lepton particle family,
and are generally thought to be elementary particles because they have no ...
s as a source of illumination. As the wavelength of an electron can be up to 100,000 times shorter than that of visible light
photon
A photon () is an elementary particle that is a quantum of the electromagnetic field, including electromagnetic radiation such as light and radio waves, and the force carrier for the electromagnetic force. Photons are massless, so they a ...
s, electron microscopes have a higher
resolving power than
light microscopes and can reveal the structure of smaller objects. A
scanning transmission electron microscope
A scanning transmission electron microscope (STEM) is a type of transmission electron microscope (TEM). Pronunciation is tɛmor sti:i:ɛm As with a conventional transmission electron microscope (CTEM), images are formed by electrons passing ...
has achieved better than 50
pm resolution in
annular dark-field imaging
Annular dark-field imaging is a method of mapping samples in a scanning transmission electron microscope (STEM). These images are formed by collecting scattered electrons with an annular dark-field detector.
Conventional TEM dark-field imaging ...
mode
and
magnifications of up to about 10,000,000× whereas most
light microscopes are limited by
diffraction to about 200
nm resolution and useful magnifications below 2000×.
Electron microscopes use shaped magnetic fields to form
electron optical lens systems that are analogous to the glass lenses of an optical light microscope.
Electron microscopes are used to investigate the
ultrastructure
Ultrastructure (or ultra-structure) is the architecture of cells and biomaterials that is visible at higher magnifications than found on a standard optical light microscope. This traditionally meant the resolution and magnification range of a co ...
of a wide range of biological and inorganic specimens including
microorganisms,
cells, large
molecules,
biopsy
A biopsy is a medical test commonly performed by a surgeon, interventional radiologist, or an interventional cardiologist. The process involves extraction of sample cells or tissues for examination to determine the presence or extent of a dise ...
samples,
metals
A metal (from Greek μέταλλον ''métallon'', "mine, quarry, metal") is a material that, when freshly prepared, polished, or fractured, shows a lustrous appearance, and conducts electricity and heat relatively well. Metals are typical ...
, and
crystals
A crystal or crystalline solid is a solid material whose constituents (such as atoms, molecules, or ions) are arranged in a highly ordered microscopic structure, forming a crystal lattice that extends in all directions. In addition, macros ...
. Industrially, electron microscopes are often used for quality control and
failure analysis. Modern electron microscopes produce electron
micrograph
A micrograph or photomicrograph is a photograph or digital image taken through a microscope or similar device to show a magnified image of an object. This is opposed to a macrograph or photomacrograph, an image which is also taken on a m ...
s using specialized digital cameras and
frame grabber
A frame grabber is an electronic device that captures (i.e., "grabs") individual, digital still frames from an analog video signal or a digital video stream. It is usually employed as a component of a computer vision system, in which video fram ...
s to capture the images.
History
![Electron Interaction with Matter](https://upload.wikimedia.org/wikipedia/commons/4/49/Electron_Interaction_with_Matter.svg)
In 1926,
Hans Busch
Hans Walter Hugo Busch (27 February 1884 in Jüchen, North Rhine-Westphalia, Germany – 16 February 1973 in Darmstadt, Hesse) was a German physicist. He was a pioneer of electron optics and laid the theoretical basis for the electron microscop ...
developed the electromagnetic lens.
According to
Dennis Gabor
Dennis Gabor ( ; hu, Gábor Dénes, ; 5 June 1900 – 9 February 1979) was a Hungarian-British electrical engineer and physicist, most notable for inventing holography, for which he later received the 1971 Nobel Prize in Physics. He obtained ...
, the physicist
Leó Szilárd
Leo Szilard (; hu, Szilárd Leó, pronounced ; born Leó Spitz; February 11, 1898 – May 30, 1964) was a Hungarian-German-American physicist and inventor. He conceived the nuclear chain reaction in 1933, patented the idea of a nuclear ...
tried in 1928 to convince him to build an electron microscope, for which he had filed a patent. The first prototype electron microscope, capable of four-hundred-power magnification, was developed in 1931 by the physicist
Ernst Ruska
Ernst August Friedrich Ruska (; 25 December 1906 – 27 May 1988) was a German physicist who won the Nobel Prize in Physics in 1986 for his work in electron optics, including the design of the first electron microscope.
Life and career
Erns ...
and the electrical engineer
Max Knoll
Max Knoll (17 July 1897 – 6 November 1969) 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 T ...
at the Berlin Technische Hochschule or Berlin Technical University. The apparatus was the first practical demonstration of the principles of electron microscopy.
In May of the same year,
Reinhold Rudenberg, the scientific director of
Siemens-Schuckert
Siemens-Schuckert (or Siemens-Schuckertwerke) was a German electrical engineering company headquartered in Berlin, Erlangen and Nuremberg that was incorporated into the Siemens AG in 1966.
Siemens Schuckert was founded in 1903 when Siemens & H ...
werke, obtained a patent for an electron microscope. In 1932, Ernst Lubcke of
Siemens & Halske
Siemens & Halske AG (or Siemens-Halske) was a German electrical engineering company that later became part of Siemens.
It was founded on 12 October 1847 as ''Telegraphen-Bauanstalt von Siemens & Halske'' by Werner von Siemens and Johann Ge ...
built and obtained images from a prototype electron microscope, applying the concepts described in Rudenberg's patent.
In the following year, 1933, Ruska built the first electron microscope that exceeded the resolution attainable with an optical (light) microscope.
Four years later, in 1937, Siemens financed the work of Ernst Ruska and
Bodo von Borries
Bodo von Borries (born 22 May 1905 in Herford, North Rhine-Westphalia, Germany — died 17 July 1956 in Aachen, North Rhine-Westphalia) was a German physicist. He was the co-inventor of the electron microscope.
Von Borries studied electrical en ...
, and employed
Helmut Ruska
Helmut Ruska (June 7, 1908, Heidelberg - August 30, 1973) 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. During this time, ...
, Ernst's brother, to develop applications for the microscope, especially with biological specimens.
Also in 1937,
Manfred von Ardenne
Manfred von Ardenne (20 January 1907 – 26 May 1997) was a German researcher and applied physicist and inventor. He took out approximately 600 patents in fields including electron microscopy, medical technology, nuclear technology, plasma physics ...
pioneered the
scanning electron microscope. Siemens produced the first commercial electron microscope in 1938. The first North American electron microscopes were constructed in the 1930, at the
Washington State University
Washington State University (Washington State, WSU, or informally Wazzu) is a public land-grant research university with its flagship, and oldest, campus in Pullman, Washington. Founded in 1890, WSU is also one of the oldest land-grant uni ...
by Anderson and Fitzsimmons and the
University of Toronto
The University of Toronto (UToronto or U of T) is a public university, public research university in Toronto, Ontario, Canada, located on the grounds that surround Queen's Park (Toronto), Queen's Park. It was founded by royal charter in 1827 ...
, by
Eli Franklin Burton
Eli Franklin Burton, (February 14, 1879 – July 6, 1948) 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 ...
and students Cecil Hall,
James Hillier
James Hillier, (August 22, 1915 – January 15, 2007) was a Canadian-American scientist and inventor who designed and built, with Albert Prebus, the first successful high-resolution electron microscope in North America in 1938.
Biography
B ...
, and Albert Prebus. Siemens produced a transmission electron microscope (TEM) in 1939. Although current transmission electron microscopes are capable of two million-power magnification, as scientific instruments, they remain based upon Ruska's
prototype.
Types
Transmission electron microscope (TEM)
The original form of the electron microscope, the
transmission electron microscope
Transmission electron microscopy (TEM) is a microscopy technique in which a beam of electrons is transmitted through a specimen to form an image. The specimen is most often an ultrathin section less than 100 nm thick or a suspension on a gr ...
(TEM), uses a
high voltage
High voltage electricity refers to electrical potential large enough to cause injury or damage. In certain industries, ''high voltage'' refers to voltage above a certain threshold. Equipment and conductors that carry high voltage warrant sp ...
electron beam to illuminate the specimen and create an image. The electron beam is produced by an
electron gun
An electron gun (also called electron emitter) is an electrical component in some vacuum tubes that produces a narrow, collimated electron beam that has a precise kinetic energy. The largest use is in cathode-ray tubes (CRTs), used in nearly ...
, commonly fitted with a
tungsten
Tungsten, or wolfram, is a chemical element with the symbol W and atomic number 74. Tungsten is a rare metal found naturally on Earth almost exclusively as compounds with other elements. It was identified as a new element in 1781 and first isol ...
filament
cathode
A cathode is the electrode from which a conventional current leaves a polarized electrical device. This definition can be recalled by using the mnemonic ''CCD'' for ''Cathode Current Departs''. A conventional current describes the direction in wh ...
as the electron source. The electron beam is accelerated by an
anode
An anode is an electrode of a polarized electrical device through which conventional current enters the device. This contrasts with a cathode, an electrode of the device through which conventional current leaves the device. A common mnemonic ...
typically at +100 k
eV (40 to 400 keV) with respect to the cathode, focused by
electrostatic
Electrostatics is a branch of physics that studies electric charges at rest ( static electricity).
Since classical times, it has been known that some materials, such as amber, attract lightweight particles after rubbing. The Greek word for amb ...
and
electromagnetic
In physics, electromagnetism is an interaction that occurs between particles with electric charge. It is the second-strongest of the four fundamental interactions, after the strong force, and it is the dominant force in the interactions o ...
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
In optical engineering, the objective is the optical element that gathers light from the object being observed and focuses the light rays to produce a real image. Objectives can be a single lens or mirror, or combinations of several optical elem ...
system of the microscope. The spatial variation in this information (the "image") may be viewed by projecting the magnified electron image onto a fluorescent viewing screen coated with a
phosphor or
scintillator material such as
zinc sulfide
Zinc sulfide (or zinc sulphide) is an inorganic compound with the chemical formula of ZnS. This is the main form of zinc found in nature, where it mainly occurs as the mineral sphalerite. Although this mineral is usually black because of various ...
. Alternatively, the image can be photographically recorded by exposing a
photographic film or
plate
Plate may refer to:
Cooking
* Plate (dishware), a broad, mainly flat vessel commonly used to serve food
* Plates, tableware, dishes or dishware used for setting a table, serving food and dining
* Plate, the content of such a plate (for example: ...
directly to the electron beam, or a high-resolution phosphor may be coupled by means of a lens optical system or a
fibre optic light-guide to the sensor of a
digital camera
A digital camera is a camera that captures photographs in digital memory. Most cameras produced today are digital, largely replacing those that capture images on photographic film. Digital cameras are now widely incorporated into mobile devices ...
. The image detected by the digital camera may be displayed on a monitor or computer.
The resolution of TEMs is limited primarily by
spherical aberration, but a new generation of hardware correctors can reduce spherical aberration to increase the resolution in
high-resolution transmission electron microscopy
High-resolution transmission electron microscopy is an imaging mode of specialized transmission electron microscopes that allows for direct imaging of the atomic structure of samples. It is a powerful tool to study properties of materials on the a ...
(HRTEM) to below 0.5
angstrom
The angstromEntry "angstrom" in the Oxford online dictionary. Retrieved on 2019-03-02 from https://en.oxforddictionaries.com/definition/angstrom.Entry "angstrom" in the Merriam-Webster online dictionary. Retrieved on 2019-03-02 from https://www.m ...
(50
picometres),
enabling magnifications above 50 million times. The ability of HRTEM to determine the positions of atoms within materials is useful for nano-technologies research and development.
Transmission electron microscopes are often used in
electron diffraction mode. The advantages of electron diffraction over
X-ray crystallography
X-ray crystallography is the experimental science determining the atomic and molecular structure of a crystal, in which the crystalline structure causes a beam of incident X-rays to diffract into many specific directions. By measuring the angles ...
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
In physics, the phase problem is the problem of loss of information concerning the phase that can occur when making a physical measurement. The name comes from the field of X-ray crystallography, where the phase problem has to be solved for the de ...
faced by the X-ray crystallographers after obtaining their X-ray diffraction patterns.
One major disadvantage of the transmission electron microscope is the need for extremely thin sections of the specimens, typically about 100 nanometers. Creating these thin sections for biological and materials specimens is technically very challenging. Semiconductor thin sections can be made using a
focused ion beam
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. A FIB setup is a s ...
. Biological tissue specimens are chemically fixed, dehydrated and embedded in a polymer resin to stabilize them sufficiently to allow ultrathin sectioning. Sections of biological specimens, organic polymers, and similar materials may require staining with heavy atom labels in order to achieve the required image contrast.
Serial-section electron microscope (ssEM)
One application of TEM is serial-section electron microscopy (ssEM), for example in analyzing the connectivity in volumetric samples of brain tissue by imaging many thin sections in sequence. This may be achieved by introducing a milling method into the imaging pipeline, by which successive slices of a 3D volume are exposed to the beam and imaged. These methods include Serial Block Face SEM (SB-SEM) and
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. A FIB setup is a s ...
-SEM (
FIB-SEM). Pre-processing of volumes to create many slices which are imaged in an automated fashion have recently achieved high-throughput imaging of volumes up to 1 mm
3. Using this method, entire local neuronal microcircuits may be resolved, though the equipment and time requirements for this are still significant: imaging a 1 mm
3 block of brain tissue required 6 months of near-continuous imaging by six TEM running in parallel.
Scanning transmission electron microscope (STEM)
The STEM rasters a focused incident probe across a specimen that (as with the TEM) has been thinned to facilitate detection of electrons scattered ''through'' the specimen. The high resolution of the TEM is thus possible in STEM. The focusing action (and aberrations) occur before the electrons hit the specimen in the STEM, but afterward in the TEM. The STEMs use of SEM-like beam rastering simplifies
annular dark-field imaging
Annular dark-field imaging is a method of mapping samples in a scanning transmission electron microscope (STEM). These images are formed by collecting scattered electrons with an annular dark-field detector.
Conventional TEM dark-field imaging ...
, and other analytical techniques, but also means that image data is acquired in serial rather than in parallel fashion. Often TEM can be equipped with the scanning option and then it can function both as TEM and STEM.
Scanning electron microscope (SEM)
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
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 ...
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 and high-energy backscattered electrons, light emission (
cathodoluminescence
Cathodoluminescence is an optical and electromagnetic phenomenon in which electrons impacting on a luminescent material such as a phosphor, cause the emission of photons which may have wavelengths in the visible spectrum. A familiar example is ...
) or
X-ray
An X-ray, or, much less commonly, X-radiation, is a penetrating form of high-energy electromagnetic radiation. Most X-rays have a wavelength ranging from 10 picometers to 10 nanometers, corresponding to frequencies in the range 30&nb ...
emission, all of which provide signals carrying information about the properties of the specimen surface, such as its topography and composition. The image displayed by an SEM maps the varying intensity of any of these signals into the image in a position corresponding to the position of the beam on the specimen when the signal was generated. In the SEM image of an ant shown below and to the right, the image was constructed from signals produced by a secondary electron detector, the normal or conventional imaging mode in most SEMs.
Generally, the image resolution of an SEM is lower than that of a TEM. However, because the SEM images the surface of a sample rather than its interior, the electrons do not have to travel through the sample. This reduces the need for extensive sample preparation to thin the specimen to electron transparency. The SEM is able to image bulk samples that can fit on its stage and still be maneuvered, including a height less than the working distance being used, often 4 millimeters for high-resolution images. The SEM also has a great depth of field, and so can produce images that are good representations of the three-dimensional surface shape of the sample. Another advantage of SEMs comes with
environmental scanning electron microscope
The environmental scanning electron microscope (ESEM) is a scanning electron microscope (SEM) that allows for the option of collecting electron micrographs of specimens that are wet, uncoated, or both by allowing for a gaseous environment in ...
s (ESEM) that can produce images of good quality and resolution with hydrated samples or in low, rather than high, vacuum or under chamber gases. This facilitates imaging unfixed biological samples that are unstable in the high vacuum of conventional electron microscopes.
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
Reflection high-energy electron diffraction (RHEED) 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 materia ...
(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 domain
A magnetic domain is a region within a magnetic material in which the magnetization is in a uniform direction. This means that the individual magnetic moments of the atoms are aligned with one another and they point in the same direction. When c ...
s.
Scanning tunneling microscopy (STM)
In STM, a conductive tip held at a voltage is brought near a surface, and a profile can be obtained based on the tunneling probability of an electron from the tip to the sample since it is a function of distance.
Colour
In their most common configurations, electron microscopes produce images with a single brightness value per pixel, with the results usually rendered in
greyscale
In digital photography, computer-generated imagery, and colorimetry, a grayscale image is one in which the value of each pixel is a single sample representing only an ''amount'' of light; that is, it carries only intensity information. Gray ...
. However, often these images are then colourized through the use of feature-detection software, or simply by hand-editing using a graphics editor. This may be done to clarify structure or for aesthetic effect and generally does not add new information about the specimen.
In some configurations information about several specimen properties is gathered per pixel, usually by the use of multiple detectors.
In SEM, the attributes of topography and material contrast can be obtained by a pair of backscattered electron detectors and such attributes can be superimposed in a single colour image by assigning a different primary colour to each attribute.
Similarly, a combination of backscattered and secondary electron signals can be assigned to different colours and superimposed on a single colour micrograph displaying simultaneously the properties of the specimen.
Some types of detectors used in SEM have analytical capabilities, and can provide several items of data at each pixel. Examples are the
energy-dispersive X-ray spectroscopy
Energy-dispersive X-ray spectroscopy (EDS, EDX, EDXS or XEDS), sometimes called energy dispersive X-ray analysis (EDXA or EDAX) or energy dispersive X-ray microanalysis (EDXMA), is an analytical technique used for the elemental analysis or chemi ...
(EDS) detectors used in elemental analysis and
cathodoluminescence microscope
A cathodoluminescence (CL) microscope combines methods from electron and regular (light optical) microscopes. It is designed to study the luminescence characteristics of polished thin sections of solids irradiated by an electron beam.
Using a ca ...
(CL) systems that analyse the intensity and spectrum of electron-induced
luminescence
Luminescence is spontaneous emission of light by a substance not resulting from heat; or "cold light".
It is thus a form of cold-body radiation. It can be caused by chemical reactions, electrical energy, subatomic motions or stress on a crys ...
in (for example) geological specimens. In SEM systems using these detectors, it is common to colour code the signals and superimpose them in a single colour image, so that differences in the distribution of the various components of the specimen can be seen clearly and compared. Optionally, the standard secondary electron image can be merged with the one or more compositional channels, so that the specimen's structure and composition can be compared. Such images can be made while maintaining the full integrity of the original signal, which is not modified in any way.
Sample preparation
![Golden insect 01 Pengo](https://upload.wikimedia.org/wikipedia/commons/c/ca/Golden_insect_01_Pengo.jpg)
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
protein
Proteins are large biomolecules and macromolecules that comprise one or more long chains of amino acid residues. Proteins perform a vast array of functions within organisms, including catalysing metabolic reactions, DNA replication, res ...
s with
aldehyde
In organic chemistry, an aldehyde () is an organic compound containing a functional group with the structure . The functional group itself (without the "R" side chain) can be referred to as an aldehyde but can also be classified as a formyl grou ...
s such as
formaldehyde
Formaldehyde ( , ) (systematic name methanal) is a naturally occurring organic compound with the formula and structure . The pure compound is a pungent, colourless gas that polymerises spontaneously into paraformaldehyde (refer to section ...
and
glutaraldehyde
Glutaraldehyde is an organic compound with the formula . The molecule consists of a five carbon chain doubly terminated with formyl (CHO) groups. It is usually used as a solution in water, and such solutions exists as a collection of hydrates, c ...
, and
lipid
Lipids are a broad group of naturally-occurring molecules which includes fats, waxes, sterols, fat-soluble vitamins (such as vitamins A, D, E and K), monoglycerides, diglycerides, phospholipids, and others. The functions of lipids includ ...
s with
osmium tetroxide
Osmium tetroxide (also osmium(VIII) oxide) is the chemical compound with the formula OsO4. The compound is noteworthy for its many uses, despite its toxicity and the rarity of osmium. It also has a number of unusual properties, one being that the ...
.
* ''
Negative stain In microscopy, negative staining is an established method, often used in diagnostic microscopy, for contrasting a thin specimen with an optically opaque fluid. In this technique, the background is stained, leaving the actual specimen untouched, and ...
'' – suspensions containing nanoparticles or fine biological material (such as viruses and bacteria) are briefly mixed with a dilute solution of an electron-opaque solution such as ammonium molybdate, uranyl acetate (or formate), or phosphotungstic acid. This mixture is applied to a suitably coated EM grid, blotted, then allowed to dry. Viewing of this preparation in the TEM should be carried out without delay for best results. The method is important in microbiology for fast but crude morphological identification, but can also be used as the basis for high-resolution 3D reconstruction using EM tomography methodology when carbon films are used for support. Negative staining is also used for observation of nanoparticles.
* ''
Cryofixation Cryofixation is a technique for fixation or stabilisation of biological materials as the first step in specimen preparation for electron microscopy and cryo-electron microscopy. Typical specimens for cryofixation include small samples of plant or an ...
'' – freezing a specimen so rapidly, in liquid
ethane
Ethane ( , ) is an organic chemical compound with chemical formula . At standard temperature and pressure, ethane is a colorless, odorless gas. Like many hydrocarbons, ethane is isolated on an industrial scale from natural gas and as a petroc ...
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
Cryogenic electron microscopy (cryo-EM) is a cryomicroscopy technique applied on samples cooled to cryogenic temperatures. For biological specimens, the structure is preserved by embedding in an environment of vitreous ice. An aqueous sample s ...
has branched from this technique. With the development of
cryo-electron microscopy
Cryogenic electron microscopy (cryo-EM) is a cryomicroscopy technique applied on samples cooled to cryogenic temperatures. For biological specimens, the structure is preserved by embedding in an environment of vitreous ice. An aqueous sample s ...
of vitreous sections (CEMOVIS), it is now possible to observe samples from virtually any biological specimen close to its native state.
* ''Dehydration'' – or replacement of
water
Water (chemical formula ) is an Inorganic compound, inorganic, transparent, tasteless, odorless, and Color of water, nearly colorless chemical substance, which is the main constituent of Earth's hydrosphere and the fluids of all known living ...
with organic solvents such as
ethanol
Ethanol (abbr. EtOH; also called ethyl alcohol, grain alcohol, drinking alcohol, or simply alcohol) is an organic compound. It is an alcohol with the chemical formula . Its formula can be also written as or (an ethyl group linked to a ...
or
acetone
Acetone (2-propanone or dimethyl ketone), is an organic compound with the formula . It is the simplest and smallest ketone (). It is a colorless, highly volatile and flammable liquid with a characteristic pungent odour.
Acetone is miscib ...
, followed by
critical point drying or infiltration with embedding
resins
In polymer chemistry and materials science, resin is a solid or highly viscous substance of plant or synthetic origin that is typically convertible into polymers. Resins are usually mixtures of organic compounds. This article focuses on natu ...
. Also
freeze drying
Freeze drying, also known as lyophilization or cryodesiccation, is a low temperature dehydration process that involves freezing the product and lowering pressure, removing the ice by sublimation. This is in contrast to dehydration by most conve ...
.
* ''Embedding, biological specimens'' – after dehydration, tissue for observation in the transmission electron microscope is embedded so it can be sectioned ready for viewing. To do this the tissue is passed through a 'transition solvent' such as
propylene oxide
Propylene oxide is an acutely toxic and carcinogenic organic compound with the molecular formula CH3CHCH2O. This colourless volatile liquid with an odour similar to ether, is produced on a large scale industrially. Its major application is its us ...
(epoxypropane) or
acetone
Acetone (2-propanone or dimethyl ketone), is an organic compound with the formula . It is the simplest and smallest ketone (). It is a colorless, highly volatile and flammable liquid with a characteristic pungent odour.
Acetone is miscib ...
and then infiltrated with an
epoxy resin
In polymer chemistry and materials science, resin is a solid or highly viscous substance of plant or synthetic origin that is typically convertible into polymers. Resins are usually mixtures of organic compounds. This article focuses on n ...
such as
Araldite Araldite is a registered trademark of Huntsman Advanced Materials (previously part of Ciba-Geigy) referring to their range of engineering and structural epoxy, acrylic, and polyurethane adhesives. Swiss manufacturers originally launched Araldite D ...
, Epon, or
Durcupan Durcupan is a water-soluble epoxy resin produced by the Fluka subsidiary of Sigma-Aldrich. It is commonly used for embedding
electron microscope samples in plastic so they may be sectioned (sliced thin) with a microtome and then imaged.
Durcupa ...
; tissues may also be embedded directly in water-miscible
acrylic resin
186 px, Polyhydroxyethylmethacrylate is a typical acrylate resin.
An acrylic resin is a thermoplastic or thermosetting plastic substance typically derived from acrylic acid, methacrylic acid and acrylate monomers such as butyl acrylate and or ...
. After the resin has been polymerized (hardened) the sample is thin sectioned (ultrathin sections) and
stained – it is then ready for viewing.
* ''Embedding, materials'' – after embedding in resin, the specimen is usually ground and polished to a mirror-like finish using ultra-fine abrasives. The polishing process must be performed carefully to minimize scratches and other polishing artifacts that reduce image quality.
* ''Metal shadowing'' – Metal (e.g.
platinum
Platinum is a chemical element with the symbol Pt and atomic number 78. It is a dense, malleable, ductile, highly unreactive, precious, silverish-white transition metal. Its name originates from Spanish , a diminutive of "silver".
Pla ...
) is evaporated from an overhead electrode and applied to the surface of a biological sample at an angle. The surface topography results in variations in the thickness of the metal that are seen as variations in brightness and contrast in the electron microscope image.
* ''Replication'' – A surface shadowed with metal (e.g. platinum, or a mixture of carbon and platinum) at an angle is coated with pure carbon evaporated from carbon electrodes at right angles to the surface. This is followed by removal of the specimen material (e.g. in an acid bath, using enzymes or by mechanical separation
) to produce a surface replica that records the surface ultrastructure and can be examined using transmission electron microscopy.
* ''Sectioning'' – produces thin slices of the specimen, semitransparent to electrons. These can be cut on an
ultramicrotome with a glass or
diamond
Diamond is a solid form of the element carbon with its atoms arranged in a crystal structure called diamond cubic. Another solid form of carbon known as graphite is the chemically stable form of carbon at room temperature and pressure, ...
knife to produce ultra-thin sections about 60–90 nm thick. Disposable
glass knives are also used because they can be made in the lab and are much cheaper.
* ''
Staining'' – uses heavy metals such as
lead
Lead is a chemical element with the symbol Pb (from the Latin ) and atomic number 82. It is a heavy metal that is denser than most common materials. Lead is soft and malleable, and also has a relatively low melting point. When freshly cu ...
,
uranium
Uranium is a chemical element with the symbol U and atomic number 92. It is a silvery-grey metal in the actinide series of the periodic table. A uranium atom has 92 protons and 92 electrons, of which 6 are valence electrons. Uranium is weak ...
or
tungsten
Tungsten, or wolfram, is a chemical element with the symbol W and atomic number 74. Tungsten is a rare metal found naturally on Earth almost exclusively as compounds with other elements. It was identified as a new element in 1781 and first isol ...
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
Uranyl acetate is the acetate salt of uranium oxide, a toxic yellow-green powder useful in certain laboratory tests. Structurally, it is a coordination polymer with formula UO2(CH3CO2)2(H2O)·H2O.
Structure
In the polymer, uranyl (UO22+) ...
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.
![External face of bakers yeast membrane](https://upload.wikimedia.org/wikipedia/commons/f/ff/External_face_of_bakers_yeast_membrane.jpg)
The fresh tissue or cell suspension is frozen rapidly (cryofixation), then fractured by 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. The second coat of carbon, evaporated perpendicular to the average surface plane is often performed to improve the stability of the replica coating. The specimen is returned to room temperature and pressure, then the extremely fragile "pre-shadowed" metal replica of the fracture surface is released from the underlying biological material by careful chemical digestion with acids,
hypochlorite solution or
SDS detergent. The still-floating replica is thoroughly washed free from residual chemicals, carefully fished up on fine grids, dried then viewed in the TEM.
* ''Freeze-fracture replica immunogold labeling (FRIL)'' – the freeze-fracture method has been modified to allow the identification of the components of the fracture face by immunogold labeling. Instead of removing all the underlying tissue of the thawed replica as the final step before viewing in the microscope the tissue thickness is minimized during or after the fracture process. The thin layer of tissue remains bound to the metal replica so it can be immunogold labeled with antibodies to the structures of choice. The thin layer of the original specimen on the replica with gold attached allows the identification of structures in the fracture plane. There are also related methods which label the surface of etched cells and other replica labeling variations.
* ''Ion beam milling'' – thins samples until they are transparent to electrons by firing
ions
An ion () is an atom or molecule with a net electrical charge.
The charge of an electron is considered to be negative by convention and this charge is equal and opposite to the charge of a proton, which is considered to be positive by conven ...
(typically
argon
Argon is a chemical element with the symbol Ar and atomic number 18. It is in group 18 of the periodic table and is a noble gas. Argon is the third-most abundant gas in Earth's atmosphere, at 0.934% (9340 ppmv). It is more than twice as ...
) at the surface from an angle and sputtering material from the surface. A subclass of this is
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. A FIB setup is a s ...
milling, where
gallium ions are used to produce an electron transparent membrane in a specific region of the sample, for example through a device within a microprocessor. Ion beam milling may also be used for cross-section polishing prior to SEM analysis of materials that are difficult to prepare using mechanical polishing.
* ''Conductive coating'' – an ultrathin coating of electrically conducting material, deposited either by high vacuum evaporation or by low vacuum sputter coating of the sample. This is done to prevent the accumulation of static electric fields at the specimen due to the electron irradiation required during imaging. The coating materials include gold, gold/palladium, platinum, tungsten, graphite, etc.
* ''
Earthing'' – to avoid electrical charge accumulation on a conductively coated sample, it is usually electrically connected to the metal sample holder. Often an
electrically conductive adhesive is used for this purpose.
Disadvantages
![Jeol Transmission and scanning EM](https://upload.wikimedia.org/wikipedia/commons/3/39/Jeol_Transmission_and_scanning_EM.jpg)
Electron microscopes are expensive to build and maintain, but the capital and running costs of
confocal light microscope systems now overlaps with those of basic electron microscopes. Microscopes designed to achieve high resolutions must be housed in stable buildings (sometimes underground) with special services such as magnetic field canceling systems.
The samples largely have to be viewed in
vacuum
A vacuum is a space devoid of matter. The word is derived from the Latin adjective ''vacuus'' for "vacant" or " void". An approximation to such vacuum is a region with a gaseous pressure much less than atmospheric pressure. Physicists often di ...
, as the molecules that make up air would scatter the electrons. An exception is
liquid-phase electron microscopy
Liquid-phase electron microscopy (LP EM) refers to a class of methods for imaging specimens in liquid with nanometer spatial resolution using electron microscopy. LP-EM overcomes the key limitation of electron microscopy: since the electron opti ...
using either a closed liquid cell or an environmental chamber, for example, in the
environmental scanning electron microscope
The environmental scanning electron microscope (ESEM) is a scanning electron microscope (SEM) that allows for the option of collecting electron micrographs of specimens that are wet, uncoated, or both by allowing for a gaseous environment in ...
, which allows hydrated samples to be viewed in a low-pressure (up to ) wet environment. Various techniques for
in situ electron microscopy of gaseous samples have been developed as well.
Scanning electron microscopes operating in conventional high-vacuum mode usually image conductive specimens; therefore non-conductive materials require conductive coating (gold/palladium alloy, carbon, osmium, etc.). The low-voltage mode of modern microscopes makes possible the observation of non-conductive specimens without coating. Non-conductive materials can be imaged also by a variable pressure (or environmental) scanning electron microscope.
Small, stable specimens such as
carbon nanotubes
A scanning tunneling microscopy image of a single-walled carbon nanotube
Rotating single-walled zigzag carbon nanotube
A carbon nanotube (CNT) is a tube made of carbon with diameters typically measured in nanometers.
''Single-wall carbon na ...
,
diatom frustules and small mineral crystals (asbestos fibres, for example) require no special treatment before being examined in the electron microscope. Samples of hydrated materials, including almost all biological specimens, have to be prepared in various ways to stabilize them, reduce their thickness (ultrathin sectioning) and increase their electron optical contrast (staining). These processes may result in ''
artifacts'', but these can usually be identified by comparing the results obtained by using radically different specimen preparation methods. Since the 1980s, analysis of
cryofixed, vitrified specimens has also become increasingly used by scientists, further confirming the validity of this technique.
Applications
;Semiconductor and data storage
* Circuit edit
* Defect analysis
*
Failure analysis
Biology and life sciences
*
Cryobiology
Cryobiology is the branch of biology that studies the effects of low temperatures on living things within Earth's cryosphere or in science. The word cryobiology is derived from the Greek words κρῧος ryos "cold", βίος ios "life", and λό ...
*
Cryo-electron microscopy
Cryogenic electron microscopy (cryo-EM) is a cryomicroscopy technique applied on samples cooled to cryogenic temperatures. For biological specimens, the structure is preserved by embedding in an environment of vitreous ice. An aqueous sample s ...
*
Diagnostic electron microscopy
*
Drug research
Drug development is the process of bringing a new pharmaceutical drug to the market once a lead compound has been identified through the process of drug discovery. It includes preclinical research on microorganisms and animals, filing for regu ...
(e.g. antibiotics)
*
Electron tomography Electron tomography (ET) is a tomography technique for obtaining detailed 3D structures of sub-cellular, macro-molecular, or materials specimens. Electron tomography is an extension of traditional transmission electron microscopy and uses a trans ...
* Particle analysis
* Particle detection
* Protein localization
*
Structural biology
Structural biology is a field that is many centuries old which, and as defined by the Journal of Structural Biology, deals with structural analysis of living material (formed, composed of, and/or maintained and refined by living cells) at every le ...
* Tissue imaging
*
Toxicology
Toxicology is a scientific discipline, overlapping with biology, chemistry, pharmacology, and medicine, that involves the study of the adverse effects of chemical substances on living organisms and the practice of diagnosing and treating e ...
*
Virology
Virology is the scientific study of biological viruses. It is a subfield of microbiology that focuses on their detection, structure, classification and evolution, their methods of infection and exploitation of host cells for reproduction, th ...
(e.g.
viral load
Viral load, also known as viral burden, is a numerical expression of the quantity of virus in a given volume of fluid, including biological and environmental specimens. It is not to be confused with viral titre or viral titer, which depends on the ...
monitoring)
;Materials research
* Device testing and characterization
* Dynamic materials experiments
*
Electron beam-induced deposition Electron-beam-induced deposition (EBID) is a process of decomposing gaseous molecules by an electron beam leading to deposition of non-volatile fragments onto a nearby substrate. The electron beam is usually provided by a scanning electron microsco ...
* In-situ characterisation
* Materials qualification
* Medical research
*
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 nanoma ...
* Nanoprototyping
;Industry
*
Chemical
A chemical substance is a form of matter having constant chemical composition and characteristic properties. Some references add that chemical substance cannot be separated into its constituent elements by physical separation methods, i.e., w ...
/
Petrochemical
Petrochemicals (sometimes abbreviated as petchems) are the chemical products obtained from petroleum by refining. Some chemical compounds made from petroleum are also obtained from other fossil fuels, such as coal or natural gas, or renewable so ...
* Direct beam-writing fabrication
*
Food science
Food science is the basic science and applied science of food; its scope starts at overlap with agricultural science and nutritional science and leads through the scientific aspects of food safety and food processing, informing the development ...
*
Forensics
*
Fractography
Fractography is the study of the fracture surfaces of materials. Fractographic methods are routinely used to determine the cause of failure in engineering structures, especially in product failure and the practice of forensic engineering or fa ...
* Micro-characterization
*
Mining
Mining is the extraction of valuable minerals or other geological materials from the Earth, usually from an ore body, lode, vein, seam, reef, or placer deposit. The exploitation of these deposits for raw material is based on the economic ...
(mineral liberation analysis)
* Pharmaceutical QC
See also
*
Acronyms in microscopy
*
Electron diffraction
*
Electron energy loss spectroscopy
In electron energy loss spectroscopy (EELS) 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 pa ...
(EELS)
*
Electron microscope images
*
Energy filtered transmission electron microscopy Energy-filtered transmission electron microscopy (EFTEM) 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. The technique can be used to ...
(EFTEM)
*
Environmental scanning electron microscope
The environmental scanning electron microscope (ESEM) is a scanning electron microscope (SEM) that allows for the option of collecting electron micrographs of specimens that are wet, uncoated, or both by allowing for a gaseous environment in ...
(ESEM)
*
Field emission microscope
*
HiRISE
High Resolution Imaging Science Experiment is a camera on board the '' Mars Reconnaissance Orbiter'' which has been orbiting and studying Mars since 2006. The 65 kg (143 lb), US$40 million instrument was built under the direction ...
*
Immune electron microscopy
*
In situ electron microscopy
*
Karnovsky fixative
*
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 medi ...
*
Microscopy
*
Nanoscience
The nanoscopic scale (or nanoscale) usually refers to structures with a length scale applicable to nanotechnology, usually cited as 1–100 nanometers (nm). A nanometer is a billionth of a meter. The nanoscopic scale is (roughly speaking) a lo ...
*
Nanotechnology
*
Neutron microscope
Neutron microscopes use neutrons to create images by nuclear fission of lithium-6 using small-angle neutron scattering. Neutrons also have no electric charge, enabling them to penetrate substances to gain information about structure that is not a ...
*
Quantum microscopy
*
Scanning confocal electron microscopy Scanning confocal electron microscopy (SCEM) is an electron microscopy technique analogous to scanning confocal optical microscopy (SCOM). In this technique, the studied sample is illuminated by a focussed electron beam, as in other scanning micros ...
*
Scanning electron microscope (SEM)
*
Scanning tunneling microscope
A scanning tunneling microscope (STM) is a type of microscope used for imaging surfaces at the atomic level. Its development in 1981 earned its inventors, Gerd Binnig and Heinrich Rohrer, then at IBM Zürich, the Nobel Prize in Physics in 1986 ...
*
Surface science
*
Transmission Electron Aberration-Corrected Microscope
*
X-ray diffraction
*
X-ray microscope
An X-ray microscope uses electromagnetic radiation in the soft X-ray band to produce magnified images of objects. Since X-rays penetrate most objects, there is no need to specially prepare them for X-ray microscopy observations.
Unlike visible li ...
*
Low-energy electron microscopy
*
Hemispherical electron energy analyzer
A hemispherical electron energy analyzer or hemispherical deflection analyzer is a type of electron energy spectrometer generally used for applications where high energy resolution is needed—different varieties of electron spectroscopy such as an ...
References
External links
An Introduction to Electron Microscopy: resources for teachers and students
Cell Centered Database – Electron microscopy data high school (GCSE, A Level) resource
General
Animations and explanations of various types of microscopy including electron microscopy(Université Paris Sud)
Environmental Scanning Electron Microscopy (ESEM)ETH Zurich website graphics and images illustrating various procedures
Eva Nogales's Seminar: "Introduction to Electron Microscopy"FEI Image Contest FEI has had a microscopy image contest every year since 2008
Introduction to electron microscopyby David Szondy
Nanohedron.com image gallery images generated by electron microscopy
X-ray element analysis in electron microscopy information portal with X-ray microanalysis and EDX contents
History
John H.L. Watson's recollections at the University of Toronto when he worked with Hillier and Prebus
(Archives Center, National Museum of American History, Smithsonian Institution)
Other
The Royal Microscopical Society, Electron Microscopy Section (UK)Albert Lleal. Natural history subjects at Scanning Electron Microscope SEM
EM Images
{{Authority control
Microscopes
Accelerator physics
Anatomical pathology
Pathology
Scientific techniques
German inventions
Protein imaging
20th-century inventions