An electron microscope is a microscope that uses a beam of electrons as a source of illumination They use electron optic
Electron microscope
An electron microscope is a microscope that uses a beam of electrons as a source of illumination. They use electron optics that are analogous to the glass lenses of an optical light microscope to control the electron beam, for instance focusing them to produce magnified images or electron diffraction patterns. As the wavelength of an electron can be up to 100,000 times smaller than that of visible light, electron microscopes have a much higher resolution of about 0.1 nm, which compares to about 200 nm for light microscopes.Electron microscope may refer to:
Additional details can be found in the above links. This article contains some general information mainly about transmission electron microscopes.
History
Reproduction of an early electron microscope constructed by Ernst Ruska in the 1930s
Many developments laid the groundwork of the electron optics used in microscopes. One significant step was the work of Hertz in 1883 who made a cathode-ray tube with electrostatic and magnetic deflection, demonstrating manipulation of the direction of an electron beam. Others were focusing of the electrons by an axial magnetic field by Emil Wiechert in 1899, improved oxide-coated cathodes which produced more electrons by (Arthur Wehnelt) in 1905 and the development of the electromagnetic lens in 1926 by (Hans Busch). According to Dennis Gabor, the physicist Leó Szilárd tried in 1928 to convince him to build an electron microscope, for which Szilárd had filed a patent.
To this day the issue of who invented the transmission electron microscope is controversial. In 1928, at the Technical University of Berlin, Adolf Matthias (Professor of High Voltage Technology and Electrical Installations) appointed Max Knoll to lead a team of researchers to advance research on electron beams and cathode-ray oscilloscopes. The team consisted of several PhD students including Ernst Ruska. In 1931, Max Knoll and Ernst Ruska successfully generated magnified images of mesh grids placed over an anode aperture. The device, a replicate of which is shown in the figure, used two magnetic lenses to achieve higher magnifications, the first electron microscope. (Max Knoll died in 1969, so did not receive a share of the 1986 Nobel prize for the invention of electron microscopes.)
Apparently independent of this effort was work at (Siemens-Schuckert) by (Reinhold Rüdenberg). According to patent law (U.S. Patent No. 2058914 and 2070318, both filed in 1932), he is the inventor of the electron microscope, but it is not clear when he had a working instrument. He stated in a very brief article in 1932 that Siemens had been working on this for some years before the patents were filed in 1932, claiming that his effort was parallel to the university development. He died in 1961, so similar to Max Knoll, was not eligible for a share of the 1986 Nobel prize.
In the following year, 1933, Ruska and Knoll built the first electron microscope that exceeded the resolution of an optical (light) microscope. Four years later, in 1937, Siemens financed the work of Ernst Ruska and (Bodo von Borries), and employed (Helmut Ruska), Ernst's brother, to develop applications for the microscope, especially with biological specimens. Also in 1937, Manfred von Ardenne pioneered the scanning electron microscope. Siemens produced the first commercial electron microscope in 1938. The first North American electron microscopes were constructed in the 1930s, at the Washington State University by Anderson and Fitzsimmons and at the University of Toronto by (Eli Franklin Burton) and students Cecil Hall, (James Hillier), and Albert Prebus. Siemens produced a transmission electron microscope (TEM) in 1939. Although current transmission electron microscopes are capable of two million times magnification, as scientific instruments they remain similar but with improved optics.
In the 1940s, high-resolution electron microscopes were developed, enabling greater magnification and resolution. By 1965, (Albert Crewe) at the University of Chicago introduced the scanning transmission electron microscope, enhancing imaging capabilities. In the 1980s, the (field emission gun) was developed for electron microscopes, improving resolution and imaging quality. (FEI Company), founded in 1971, became a major manufacturer of electron microscopes. In 1991, (Tescan) was established, bringing innovation with their scanning electron microscopes (SEMs) and focused ion beam systems. The 2000s were marked by advancements in aberration-corrected electron microscopy, allowing for atomic-scale resolution.
Wavelength
Operating principle of a transmission electron microscope
The original form of the electron microscope, the transmission electron microscope (TEM), uses a high voltageelectron beam to illuminate the specimen and create an image. An electron beam is produced by an electron gun, with the electrons typically at 40 to 400 keV, focused by electromagnetic lenses, and transmitted through the specimen. When it emerges from the specimen, the electron beam carries information about the structure of the specimen that is magnified by lenses of the microscope. The spatial variation in this information (the "image") may be viewed by projecting the magnified electron image onto a detector. For example, the image may be viewed directly by an operator using a fluorescent viewing screen coated with a phosphor or scintillator material such as zinc sulfide. A high-resolution phosphor may also be coupled by means of a lens optical system or a fibre optic light-guide to the sensor of a digital camera. Direct electron detectors have no scintillator and are directly exposed to the electron beam, which addresses some of the limitations of scintillator-coupled cameras.
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 (HRTEM) to below 0.5 angstrom (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 are that the specimen need not be a single crystal or even a polycrystalline powder.[]
Scanning transmission electron microscope (STEM)
The STEM rasters a focused incident probe across a specimen. The high resolution of the TEM is thus possible in STEM. The focusing action (and aberrations) occur before the electrons hit the specimen in the STEM, but afterward in the TEM. The STEMs use of SEM-like beam rastering simplifies (annular dark-field imaging), and other analytical techniques, but also means that image data is acquired in serial rather than in parallel fashion.[]
Scanning electron microscope (SEM)
Operating principle of a scanning electron microscopeImage of Bacillus subtilis taken with a 1960s electron microscope
The SEM produces images by probing the specimen with a focused electron beam that is scanned across the specimen (raster scanning). When the electron beam interacts with the specimen, it loses energy by a variety of mechanisms. The lost energy is converted into alternative forms such as heat, emission of low-energy secondary electrons and high-energy backscattered electrons, light emission (cathodoluminescence) or X-ray emission, 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, 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 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.[]
In their most common configurations, electron microscopes produce images with a single brightness value per pixel, with the results usually rendered in greyscale. 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.
Sample preparation for TEM
An insect (coated in gold) for viewing with a scanning electron microscope
Materials to be viewed in a transmission electron microscope may require processing to produce a suitable sample. The technique required varies depending on the specimen and the analysis required:
Cryofixation – freezing a specimen so that the water forms vitreous (non-crystalline) ice. This preserves the specimen in a snapshot of its native state. Methods to achieve this vitrification include plunge freezing rapidly in liquid ethane, and high pressure freezing. An entire field called cryo-electron microscopy has branched from this technique. With the development of cryo-electron microscopy of vitreous sections (CEMOVIS) and cryo-focused ion beam milling of lamellae, it is now possible to observe samples from virtually any biological specimen close to its native state.
Dehydration – replacement of water with organic solvents such as ethanol or acetone, followed by (critical point drying) or infiltration with embedding resins. See also freeze drying.[]
Embedding, biological specimens – after dehydration, tissue for observation in the transmission electron microscope is embedded so it can be sectioned ready for viewing. To do this the tissue is passed through a 'transition solvent' such as propylene oxide (epoxypropane) or acetone and then infiltrated with an epoxyresin such as (Araldite), Epon, or (Durcupan); tissues may also be embedded directly in water-miscible acrylic resin. After the resin has been polymerized (hardened) the sample is sectioned by (ultramicrotomy) and stained.[]
Embedding, materials – after embedding in resin, the specimen is usually ground and polished to a mirror-like finish using ultra-fine abrasives.[]
Freeze-fracture or freeze-etch – a preparation method particularly useful for examining lipid membranes and their incorporated proteins in "face on" view.Freeze-fracturing helps to peel open membranes to allow visualization of what is insideExternal face of bakers yeast membrane showing the small holes where proteins are fractured out, sometimes as small ring patterns.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 (typically argon) at the surface from an angle and sputtering material from the surface. A subclass of this is focused ion beam milling, where gallium ions are used to produce an electron transparent membrane or 'lamella' in a specific region of the sample, for example through a device within a microprocessor or a focused ion beam SEM. Ion beam milling may also be used for cross-section polishing prior to analysis of materials that are difficult to prepare using mechanical polishing.[]
(Negative stain) – suspensions containing nanoparticles or fine biological material (such as viruses and bacteria) are briefly mixed with a dilute solution of an electron-opaque solution such as ammonium molybdate, uranyl acetate (or formate), or (phosphotungstic acid).[] This mixture is applied to an EM grid, pre-coated with a plastic film such as formvar, 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.[]
Sectioning – produces thin slices of the specimen, semitransparent to electrons. These can be cut using (ultramicrotomy) on an (ultramicrotome) with a glass or diamond 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. Sections can also be created in situ by milling in a focused ion beam SEM, where the section is known as a lamella.
Staining – uses heavy metals such as lead, uranium or tungsten to scatter imaging electrons and thus give contrast between different structures, since many (especially biological) materials are nearly "transparent" to electrons (weak phase objects). In biology, specimens can be stained "en bloc" before embedding and also later after sectioning. Typically thin sections are stained for several minutes with an aqueous or alcoholic solution of uranyl acetate followed by aqueous lead citrate.
EM workflows
Early electron microscopy of biological specimens was often descriptive, making use of the newly available higher resolution. This is still the case for various applications, such as (diagnostic electron microscopy).
However, electron microscopes are now frequently used in more complex workflows, with each workflow typically using multiple technologies to enable more complex and/or more quantitative analyses of a sample. A few examples are outlined below, but this should not be considered an exhaustive list. The choice of workflow will be highly dependent on the application and the requirements of the corresponding scientific questions, such as resolution, volume, nature of the target molecule, etc.
For example, images from light and electron microscopy of the same region of a sample can be overlaid to correlate the data from the two modalities. This is commonly used to provide higher resolution contextual EM information about a fluorescently labelled structure. This correlative light and electron microscopy ((CLEM)) is one of a range of correlative workflows now available. Another example is high resolution mass spectrometry (ion microscopy), which has been used to provide correlative information about subcellular antibiotic localisation, data that would be difficult to obtain by other means.[]
The initial role of electron microscopes in imaging two-dimensional slices (TEM) or a specimen surface (SEM with secondary electrons) has also increasingly expanded into the depth of samples. An early example of these ‘’ workflows was simply to stack TEM images of serial sections cut through a sample. The next development was virtual reconstruction of a thick section (200-500 nm) volume by backprojection of a set of images taken at different tilt angles - TEM tomography.
Serial imaging for volume EM
To acquire datasets of larger depths than TEM tomography (micrometers or millimeters in the z axis), a series of images taken through the sample depth can be used. For example, ribbons of serial sections can be imaged in a TEM as described above, and when thicker sections are used, serial TEM tomography can be used to increase the z-resolution. More recently, back scattered electron (BSE) images can be acquired of a larger series of sections collected on silicon wafers, known as SEM array tomography. An alternative approach is to use BSE SEM to image the block surface instead of the section, after each section has been removed. By this method, an ultramicrotome installed in an SEM chamber can increase automation of the workflow; the specimen block is loaded in the chamber and the system programmed to continuously cut and image through the sample. This is known as serial block face SEM. A related method uses focused ion beam milling instead of an ultramicrotome to remove sections. In these serial imaging methods, the output is essentially a sequence of images through a specimen block that can be digitally aligned in sequence and thus reconstructed into a dataset. The increased volume available in these methods has expanded the capability of electron microscopy to address new questions, such as mapping neural connectivity in the brain, and membrane contact sites between organelles.
Disadvantages
(JEOL) transmission and scanning electron microscope made in the mid-1970s
Electron microscopes are expensive to build and maintain. 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, as the molecules that make up air would scatter the electrons. An exception is (liquid-phase electron microscopy) using either a closed liquid cell or an environmental chamber, for example, in the (environmental scanning electron microscope), which allows hydrated samples to be viewed in a low-pressure (up to 20 Torr or 2.7 kPa) wet environment. Various techniques for (in situ electron microscopy) of gaseous samples have been developed.
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, 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.
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An electron microscope is a microscope that uses a beam of electrons as a source of illumination They use electron optics that are analogous to the glass lenses of an optical light microscope to control the electron beam for instance focusing them to produce magnified images or electron diffraction patterns As the wavelength of an electron can be up to 100 000 times smaller than that of visible light electron microscopes have a much higher resolution of about 0 1 nm which compares to about 200 nm for light microscopes Electron microscope may refer to Transmission electron microscopy TEM where swift electrons go through a thin sample Scanning transmission electron microscopy STEM which is similar to TEM with a scanned electron probe Scanning electron microscope SEM which is similar to STEM but with thick samples Electron microprobe similar to a SEM but more for chemical analysis Ultrafast scanning electron microscopy version of a SEM that can operate very fast Low energy electron microscopy LEEM used to image surfaces Photoemission electron microscopy PEEM which is similar to LEEM using electrons emitted from surfaces by photonsA transmission electron microscope from 2002An image of an ant in a scanning electron microscopeThis article needs additional citations for verification Please help improve this article by adding citations to reliable sources Unsourced material may be challenged and removed Find sources Electron microscope news newspapers books scholar JSTOR September 2023 Learn how and when to remove this message Additional details can be found in the above links This article contains some general information mainly about transmission electron microscopes HistoryReproduction of an early electron microscope constructed by Ernst Ruska in the 1930s Many developments laid the groundwork of the electron optics used in microscopes One significant step was the work of Hertz in 1883 who made a cathode ray tube with electrostatic and magnetic deflection demonstrating manipulation of the direction of an electron beam Others were focusing of the electrons by an axial magnetic field by Emil Wiechert in 1899 improved oxide coated cathodes which produced more electrons by Arthur Wehnelt in 1905 and the development of the electromagnetic lens in 1926 by Hans Busch According to Dennis Gabor the physicist Leo Szilard tried in 1928 to convince him to build an electron microscope for which Szilard had filed a patent To this day the issue of who invented the transmission electron microscope is controversial In 1928 at the Technical University of Berlin Adolf Matthias Professor of High Voltage Technology and Electrical Installations appointed Max Knoll to lead a team of researchers to advance research on electron beams and cathode ray oscilloscopes The team consisted of several PhD students including Ernst Ruska In 1931 Max Knoll and Ernst Ruska successfully generated magnified images of mesh grids placed over an anode aperture The device a replicate of which is shown in the figure used two magnetic lenses to achieve higher magnifications the first electron microscope Max Knoll died in 1969 so did not receive a share of the 1986 Nobel prize for the invention of electron microscopes Apparently independent of this effort was work at Siemens Schuckert by Reinhold Rudenberg According to patent law U S Patent No 2058914 and 2070318 both filed in 1932 he is the inventor of the electron microscope but it is not clear when he had a working instrument He stated in a very brief article in 1932 that Siemens had been working on this for some years before the patents were filed in 1932 claiming that his effort was parallel to the university development He died in 1961 so similar to Max Knoll was not eligible for a share of the 1986 Nobel prize In the following year 1933 Ruska and Knoll built the first electron microscope that exceeded the resolution of an optical light microscope Four years later in 1937 Siemens financed the work of Ernst Ruska and Bodo von Borries and employed Helmut Ruska Ernst s brother to develop applications for the microscope especially with biological specimens Also in 1937 Manfred von Ardenne pioneered the scanning electron microscope Siemens produced the first commercial electron microscope in 1938 The first North American electron microscopes were constructed in the 1930s at the Washington State University by Anderson and Fitzsimmons and at the University of Toronto by Eli Franklin Burton and students Cecil Hall James Hillier and Albert Prebus Siemens produced a transmission electron microscope TEM in 1939 Although current transmission electron microscopes are capable of two million times magnification as scientific instruments they remain similar but with improved optics In the 1940s high resolution electron microscopes were developed enabling greater magnification and resolution By 1965 Albert Crewe at the University of Chicago introduced the scanning transmission electron microscope enhancing imaging capabilities In the 1980s the field emission gun was developed for electron microscopes improving resolution and imaging quality FEI Company founded in 1971 became a major manufacturer of electron microscopes In 1991 Tescan was established bringing innovation with their scanning electron microscopes SEMs and focused ion beam systems The 2000s were marked by advancements in aberration corrected electron microscopy allowing for atomic scale resolution Wavelength source source source source source source source source Operating principle of a transmission electron microscope In a typical electron gun individual electrons which have an elementary charge e displaystyle e about 1 6 10 19 displaystyle 1 6 times 10 19 coulombs and a mass m displaystyle m about 9 1 10 31 displaystyle 9 1 times 10 31 kg with a potential of V displaystyle V volts have an energy amount of e V displaystyle e cdot V joules The wavelength is l hceV 2mc2 eV displaystyle lambda frac hc sqrt eV 2mc 2 eV where c displaystyle c is the speed of light in vacuum about c 3 108 displaystyle c 3 times 10 8 m s See electron diffraction for a full explanation TypesTransmission electron microscope TEM Diagram of a transmission electron microscope The original form of the electron microscope the transmission electron microscope TEM uses a high voltage electron beam to illuminate the specimen and create an image An electron beam is produced by an electron gun with the electrons typically at 40 to 400 keV focused by electromagnetic lenses and transmitted through the specimen When it emerges from the specimen the electron beam carries information about the structure of the specimen that is magnified by lenses of the microscope The spatial variation in this information the image may be viewed by projecting the magnified electron image onto a detector For example the image may be viewed directly by an operator using a fluorescent viewing screen coated with a phosphor or scintillator material such as zinc sulfide A high resolution phosphor may also be coupled by means of a lens optical system or a fibre optic light guide to the sensor of a digital camera Direct electron detectors have no scintillator and are directly exposed to the electron beam which addresses some of the limitations of scintillator coupled cameras 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 HRTEM to below 0 5 angstrom 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 are that the specimen need not be a single crystal or even a polycrystalline powder citation needed Scanning transmission electron microscope STEM The STEM rasters a focused incident probe across a specimen The high resolution of the TEM is thus possible in STEM The focusing action and aberrations occur before the electrons hit the specimen in the STEM but afterward in the TEM The STEMs use of SEM like beam rastering simplifies annular dark field imaging and other analytical techniques but also means that image data is acquired in serial rather than in parallel fashion citation needed Scanning electron microscope SEM source source source source source source source source Operating principle of a scanning electron microscope Image of Bacillus subtilis taken with a 1960s electron microscope The SEM produces images by probing the specimen with a focused electron beam that is scanned across the specimen raster scanning When the electron beam interacts with the specimen it loses energy by a variety of mechanisms The lost energy is converted into alternative forms such as heat emission of low energy secondary electrons and high energy backscattered electrons light emission cathodoluminescence or X ray emission all of which provide signals carrying information about the properties of the specimen surface such as its topography and composition citation needed 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 the image was constructed from signals produced by a secondary electron detector the normal or conventional imaging mode in most SEMs citation needed 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 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 citation needed In their most common configurations electron microscopes produce images with a single brightness value per pixel with the results usually rendered in greyscale 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 Sample preparation for TEMAn insect coated in gold for viewing with a scanning electron microscope Materials to be viewed in a transmission 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 this aims to stabilize the specimen s mobile macromolecular structure by chemical crosslinking of proteins with aldehydes such as formaldehyde and glutaraldehyde and lipids with osmium tetroxide Cryofixation freezing a specimen so that the water forms vitreous non crystalline ice This preserves the specimen in a snapshot of its native state Methods to achieve this vitrification include plunge freezing rapidly in liquid ethane and high pressure freezing An entire field called cryo electron microscopy has branched from this technique With the development of cryo electron microscopy of vitreous sections CEMOVIS and cryo focused ion beam milling of lamellae it is now possible to observe samples from virtually any biological specimen close to its native state Dehydration replacement of water with organic solvents such as ethanol or acetone followed by critical point drying or infiltration with embedding resins See also freeze drying citation needed Embedding biological specimens after dehydration tissue for observation in the transmission electron microscope is embedded so it can be sectioned ready for viewing To do this the tissue is passed through a transition solvent such as propylene oxide epoxypropane or acetone and then infiltrated with an epoxy resin such as Araldite Epon or Durcupan tissues may also be embedded directly in water miscible acrylic resin After the resin has been polymerized hardened the sample is sectioned by ultramicrotomy and stained citation needed Embedding materials after embedding in resin the specimen is usually ground and polished to a mirror like finish using ultra fine abrasives citation needed Freeze fracture or freeze etch a preparation method particularly useful for examining lipid membranes and their incorporated proteins in face on view Freeze fracturing helps to peel open membranes to allow visualization of what is insideExternal face of bakers yeast membrane showing the small holes where proteins are fractured out sometimes as small ring patterns 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 citation needed 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 typically argon at the surface from an angle and sputtering material from the surface A subclass of this is focused ion beam milling where gallium ions are used to produce an electron transparent membrane or lamella in a specific region of the sample for example through a device within a microprocessor or a focused ion beam SEM Ion beam milling may also be used for cross section polishing prior to analysis of materials that are difficult to prepare using mechanical polishing citation needed Negative stain suspensions containing nanoparticles or fine biological material such as viruses and bacteria are briefly mixed with a dilute solution of an electron opaque solution such as ammonium molybdate uranyl acetate or formate or phosphotungstic acid citation needed This mixture is applied to an EM grid pre coated with a plastic film such as formvar 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 citation needed Sectioning produces thin slices of the specimen semitransparent to electrons These can be cut using ultramicrotomy on an ultramicrotome with a glass or diamond 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 Sections can also be created in situ by milling in a focused ion beam SEM where the section is known as a lamella Staining uses heavy metals such as lead uranium or tungsten to scatter imaging electrons and thus give contrast between different structures since many especially biological materials are nearly transparent to electrons weak phase objects In biology specimens can be stained en bloc before embedding and also later after sectioning Typically thin sections are stained for several minutes with an aqueous or alcoholic solution of uranyl acetate followed by aqueous lead citrate EM workflowsEarly electron microscopy of biological specimens was often descriptive making use of the newly available higher resolution This is still the case for various applications such as diagnostic electron microscopy However electron microscopes are now frequently used in more complex workflows with each workflow typically using multiple technologies to enable more complex and or more quantitative analyses of a sample A few examples are outlined below but this should not be considered an exhaustive list The choice of workflow will be highly dependent on the application and the requirements of the corresponding scientific questions such as resolution volume nature of the target molecule etc For example images from light and electron microscopy of the same region of a sample can be overlaid to correlate the data from the two modalities This is commonly used to provide higher resolution contextual EM information about a fluorescently labelled structure This correlative light and electron microscopy CLEM is one of a range of correlative workflows now available Another example is high resolution mass spectrometry ion microscopy which has been used to provide correlative information about subcellular antibiotic localisation data that would be difficult to obtain by other means citation needed The initial role of electron microscopes in imaging two dimensional slices TEM or a specimen surface SEM with secondary electrons has also increasingly expanded into the depth of samples An early example of these workflows was simply to stack TEM images of serial sections cut through a sample The next development was virtual reconstruction of a thick section 200 500 nm volume by backprojection of a set of images taken at different tilt angles TEM tomography Serial imaging for volume EM To acquire datasets of larger depths than TEM tomography micrometers or millimeters in the z axis a series of images taken through the sample depth can be used For example ribbons of serial sections can be imaged in a TEM as described above and when thicker sections are used serial TEM tomography can be used to increase the z resolution More recently back scattered electron BSE images can be acquired of a larger series of sections collected on silicon wafers known as SEM array tomography An alternative approach is to use BSE SEM to image the block surface instead of the section after each section has been removed By this method an ultramicrotome installed in an SEM chamber can increase automation of the workflow the specimen block is loaded in the chamber and the system programmed to continuously cut and image through the sample This is known as serial block face SEM A related method uses focused ion beam milling instead of an ultramicrotome to remove sections In these serial imaging methods the output is essentially a sequence of images through a specimen block that can be digitally aligned in sequence and thus reconstructed into a dataset The increased volume available in these methods has expanded the capability of electron microscopy to address new questions such as mapping neural connectivity in the brain and membrane contact sites between organelles DisadvantagesJEOL transmission and scanning electron microscope made in the mid 1970s Electron microscopes are expensive to build and maintain 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 as the molecules that make up air would scatter the electrons An exception is liquid phase electron microscopy using either a closed liquid cell or an environmental chamber for example in the environmental scanning electron microscope which allows hydrated samples to be viewed in a low pressure up to 20 Torr or 2 7 kPa wet environment Various techniques for in situ electron microscopy of gaseous samples have been developed 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 citation needed Small stable specimens such as carbon nanotubes diatom frustules and small mineral crystals asbestos fibres for example require no special treatment before being examined in the electron microscope Samples of hydrated materials including almost all biological specimens have to be prepared in various ways to stabilize them reduce their thickness ultrathin sectioning and increase their electron optical contrast staining These processes may result in artifacts but these can usually be identified by comparing the results obtained by using radically different specimen preparation methods Since the 1980s analysis of cryofixed vitrified specimens has also become increasingly used by scientists further confirming the validity of this technique See alsoList of materials analysis methods Electron diffraction Electron energy loss spectroscopy EELS Electron microscope images Energy filtered transmission electron microscopy EFTEM Environmental scanning electron microscope ESEM Immune electron microscopy In situ electron microscopy Low energy electron microscopy Microscope image processing Microscopy Nanotechnology Scanning confocal electron microscopy Scanning electron microscope SEM Thin section Transmission Electron Aberration Corrected MicroscopeReferences Electron microscope Encyclopaedia Britannica Retrieved June 26 2024 Calbick C J 1944 Historical Background of Electron Optics Journal of Applied Physics 15 10 685 690 Bibcode 1944JAP 15 685C doi 10 1063 1 1707371 ISSN 0021 8979 Hertz Heinrich 2019 Introduction to Heinrich Hertz s Miscellaneous Papers 1895 by Philipp Lenard Heinrich Rudolf Hertz 1857 1894 Routledge pp 87 88 doi 10 4324 9780429198960 4 ISBN 978 0 429 19896 0 S2CID 195494352 retrieved 2023 02 24 Wiechert E 1899 Experimentelle Untersuchungen uber die Geschwindigkeit und die magnetische Ablenkbarkeit der Kathodenstrahlen Annalen der Physik und Chemie in German 305 12 739 766 Bibcode 1899AnP 305 739W doi 10 1002 andp 18993051203 Wehnelt A 1905 X On the discharge of negative ions by glowing metallic oxides and allied phenomena The London Edinburgh and Dublin Philosophical Magazine and Journal of Science 10 55 80 90 doi 10 1080 14786440509463347 ISSN 1941 5982 Busch H 1926 Berechnung der Bahn von Kathodenstrahlen im axialsymmetrischen elektromagnetischen Felde Annalen der Physik in German 386 25 974 993 Bibcode 1926AnP 386 974B doi 10 1002 andp 19263862507 Dannen Gene 1998 Leo Szilard the Inventor A Slideshow 1998 Budapest conference talk dannen com Mulvey T 1962 Origins and historical development of the electron microscope British Journal of Applied 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