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Electron microscope
Electron microscope
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A modern transmission electron microscope (TITAN)

An electron microscope is a microscope that uses a beam of electrons as a source of illumination. It uses electron optics that are analogous to the glass lenses of an optical light microscope to control the electron beam, for instance focusing it 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.[1] Electron microscope may refer to:

Additional details can be found in the above links. This article contains some general information mainly about transmission and scanning electron microscopes.

History

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See also: Transmission electron microscopy § History

Many developments laid the groundwork of the electron optics used in microscopes.[2] One significant step was the work of Hertz in 1883[3] 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,[4] improved oxide-coated cathodes which produced more electrons by Arthur Wehnelt in 1905[5] and the development of the electromagnetic lens in 1926 by Hans Busch.[6] 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.[7]

Reproduction of an early electron microscope constructed by Ernst Ruska in the 1930s

To this day the issue of who invented the transmission electron microscope is controversial.[8][9][10][11] In 1928, at the Technische Hochschule in Charlottenburg (now Technische Universität 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[12][13] 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[14] and 2070318,[15] 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[16] 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.[17]

In the following year, 1933, Ruska and Knoll built the first electron microscope that exceeded the resolution of an optical (light) microscope.[18] 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.[18][19] Also in 1937, Manfred von Ardenne pioneered the scanning electron microscope.[20] Siemens produced the first commercial electron microscope in 1938.[21] The first North American electron microscopes were constructed in the 1930s, at the Washington State University by Anderson and Fitzsimmons [22] 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.[23] 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.[24] By 1965, Albert Crewe at the University of Chicago introduced the scanning transmission electron microscope using a field emission source,[25] enabling scanning microscopes at high resolution.[26] By the early 1980s improvements in mechanical stability as well as the use of higher accelerating voltages enabled imaging of materials at the atomic scale.[27][28] In the 1980s, the field emission gun became common for electron microscopes, improving the image quality due to the additional coherence and lower chromatic aberrations. The 2000s were marked by advancements in aberration-corrected electron microscopy, allowing for significant improvements in resolution and clarity of images.[29][30]

Types of electron microscopes

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Operating principle of a transmission electron microscope

Transmission electron microscope (TEM)

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Main article: Transmission electron microscope
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 having energies in the range 20 to 400 keV, focused by electromagnetic lenses, and transmitted through a thin specimen. When it emerges from the specimen, the electron beam carries information about the structure of the specimen that is then magnified by the 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. More commonly a high-resolution phosphor is coupled by means of a lens optical system or a fibre optic light-guide to the sensor of a digital camera. A different approach is to use a direct electron detector which has no scintillator, which addresses some of the limitations of scintillator-coupled cameras.[31]

For many years the resolution of TEMs was limited by aberrations of the electron optics, primarily the spherical aberration. In most recent instruments hardware correctors can reduce spherical aberration and other aberrations, improving the resolution in high-resolution transmission electron microscopy (HRTEM) to below 0.5 angstrom (50 picometres),[32] enabling magnifications of more than 50 million times.[33] The ability of HRTEM to determine the positions of atoms within materials is useful for many areas of research and development.[34]

Scanning electron microscope (SEM)

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Operating principle of a scanning electron microscope
Main article: Scanning electron microscope

An 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 and is scattered in different directions by a variety of mechanisms. These interactions lead to, among other events, emission of low-energy secondary electrons and high-energy backscattered electrons, light emission (cathodoluminescence) or X-ray emission. All of these signals carrying information about the specimen, such as the surface topography and composition. The image displayed when using an SEM shows the variation in the intensity of any of these signals as an image. In these each position in the image corresponding to a position of the beam on the specimen when the signal was generated.[35]: 1–15 

TESCAN S8000X SEM

SEMs are different from TEMs in that they use electrons with much lower energy, generally below 20 keV,[36] while TEMs generally use electrons with energies in the range of 80-300 keV.[37] Thus, the electron sources and optics of the two microscopes have different designs, and they are normally separate instruments.[38]

Scanning transmission electron microscope (STEM)

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Main article: Scanning transmission electron microscopy

A STEM combines features of both a TEM and a SEM by rastering a focused incident probe across a specimen, but now mainly using the electrons which are transmitted through the sample. Many types of imaging are common to both TEM and STEM, but some such as annular dark-field imaging and other analytical techniques are much easier to perform with higher spatial resolutions in a STEM instrument. One drawback is that image data is acquired in serial rather than in parallel fashion.[35]: 75–138 

Main operating modes

[edit]
An image of an ant in an SEM

The most common methods of obtaining images in an electron microscope involve selecting different directions for the electrons that have been transmitted through a sample, and/or electrons of different energies. There are a very large number of methods of doing this, although not all are very common.

Secondary electrons

[edit]
Electron–matter interaction volume and types of signal generated in a SEM

In a SEM the signals result from interactions of the electron beam with atoms within the sample. The most common mode is to use the secondary electrons (SE) to produce images. Secondary electrons have very low energies, on the order of 50 eV, which limits their mean free path in solid matter to a few nanometers below the sample surface.[39] The electrons are detected by an Everhart–Thornley detector,[40] which is a type of collector-scintillator-photomultiplier system. The signal from secondary electrons tends to be highly localized at the point of impact of the primary electron beam, making it possible to collect images of the sample surface with a resolution of better than 1 nm, and with specialized instruments at the atomic scale.[41]

The brightness of the signal depends on the number of secondary electrons reaching the detector. If the beam enters the sample perpendicular to the surface, then the electrons come out symmetrically about the axis of the beam. As the angle of incidence increases, the interaction volume from which they cone increases and the "escape" distance from one side of the beam decreases, resulting in more secondary electrons being emitted from the sample. Thus steep surfaces and edges tend to be brighter than flat surfaces, which results in images with a well-defined, three-dimensional appearance that is similar to a reflected light image.[39]

Backscattered electrons

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Backscattered electrons (BSE) are those emitted back out from the specimen due to beam-specimen interactions where the electrons undergo elastic and inelastic scattering. They are conventionally defined as having energies from 50 eV up to the energy of the primary beam. Backscattered electrons can be used for both imaging and to form an electron backscatter diffraction (EBSD) image, the latter can be used to determine the crystallography of the specimen.[39]

Electron backscatter diffraction pattern for (001) single crystal silicon crystals taken at 20kV using Oxford S2 detector

Heavy elements (high atomic number) backscatter electrons more strongly than light elements (low atomic number), and thus appear brighter in the image, BSE images can therefore be used to detect areas with different chemical compositions.[39] To optimize the signal, dedicated backscattered electron detectors are positioned above the sample in a "doughnut" type arrangement, concentric with the electron beam, maximizing the solid angle of collection. BSE detectors are usually either scintillator or semiconductor types. When all parts of the detector are used to collect electrons symmetrically about the beam, atomic number contrast is produced. However, strong topographic contrast is produced by collecting back-scattered electrons from one side above the specimen using an asymmetrical, directional BSE detector; the resulting contrast appears as if there was illumination of the topography from that side. Semiconductor detectors can be made in radial segments that can be switched in or out to control the type of contrast produced and its directionality.[39]

Diffraction contrast imaging

[edit]

Diffraction contrast uses the variation in either or both the direction of diffracted electrons or their amplitude as a function of position as the contrast mechanism. It is one of the simplest ways to image in a transmission electron microscope, and widely used.

The idea is to use an objective aperture below the sample and select only one or a range of different diffracted directions, then use these to form an image. When the aperture includes the incident beam direction the images are called bright field, since in the absence of any sample the field of view would be uniformly bright. When the aperture excludes the incident beam the images are called dark field, since similarly without a sample the image would be uniformly dark.[42][43] One variant of this is called weak-beam dark-field microscopy, and can be used to obtain high resolution images of defects such as dislocations.[44]

High resolution imaging

[edit]
Main articles: High-resolution transmission electron microscopy and Annular dark-field imaging
CuTe High resolution image

In high-resolution transmission electron microscopy (also sometimes called high-resolution electron microscopy) a number of different diffracted beams are allowed through the objective aperture. These interfere, leading to images which represent the atomic structure of the material. These can include the incident beam direction, or with scanning transmission electron microscopes they typically are for a range of diffracted beams excluding the incident beam.[28] Depending upon how thick the samples are and the aberrations of the microscope, these images can either be directly interpreted in terms of the positions of columns of atoms, or require a more careful analysis using calculations of the multiple scattering of the electrons[45] and the effect of the contrast transfer function of the microscope.[46]

Main articles: Electron holography and 4D STEM

There are many other imaging variants that can also to lead to atomic level information. Electron holography uses the interference of electrons which have been through the sample and a reference beam.[47] 4D STEM collects diffraction data at each point using a scanning instrument, then processes them to produce different types of images.[48]

X-ray microanalysis

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Main article: Energy-dispersive X-ray spectroscopy
EDS spectrum of the mineral crust of the vent shrimp Rimicaris exoculata[49] Most of these peaks are K-alpha and K-beta lines. One peak is from the L shell of iron.

X-ray microanalysis is a method of obtaining local chemical information within electron microscopes of all types, although it is most commonly used in scanning instruments. When high energy electrons interact with atoms they can knock out electrons, particularly those in the inner shells and core electrons. These are then filled by valence electron, and the energy difference between the valence and core states can be converted into an x-ray which is detected by a spectrometer. The energies of these x-rays is somewhat specific to the atomic species, so local chemistry can be probed.[39]

EELS

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Main article: Electron energy loss spectroscopy
Experimental electron energy loss spectrum, showing the major features: zero-loss peak, plasmon peaks and core loss edge.

Similar to X-ray microanalysis, the energies of electrons which have transmitted through a sample can be analyzed and yield information ranging from details of the local electronic structure to chemical information.[50]

Electron diffraction

[edit]
Main article: Electron diffraction

Transmission electron microscopes can be used in electron diffraction mode where a map of the angles of the electrons leaving the sample is produced. The advantages of electron diffraction over X-ray crystallography are primarily in the size of the crystals. In X-ray crystallography, crystals are commonly visible by the naked eye and are generally in the hundreds of micrometers in length. In comparison, crystals for electron diffraction must be less than a few hundred nanometers in thickness, and have no lower boundary of size. Additionally, electron diffraction is done on a TEM, which can also be used to obtain other types of information, rather than requiring a separate instrument.[51][37]

Variations in CBED with thickness for Si (001)
Main articles: Convergent beam electron diffraction and Precession electron diffraction

There are many variants on electron diffraction, depending upon exactly what type of illumination conditions are used. If a parallel beam is used with an aperture to limit the region exposed to the electrons then sharp diffraction features are normally observed, a technique called selected area electron diffraction. This is often the main technique used. Another common approach uses conical illumination and is called convergent beam electron diffraction (CBED). This is good for determining the symmetry of materials. A third is precession electron diffraction, where a parallel beam is spun around a large angle, producing a type of average diffraction pattern.[52] These often have less multiple scattering.[53]

Other electron microscope techniques

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Aberration corrected instruments

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Scanning transmission electron microscope equipped with a 3rd-order spherical aberration corrector
Main article: Aberration-corrected transmission electron microscopy

Aberration-corrected transmission electron microscopy (AC-TEM) is the general term for electron microscopes where electro optical components are introduced to reduce the aberrations that would otherwise limit the resolution of the images. Historically electron microscopes had quite severe aberrations, and until about the start of the 21st century the resolution was limited, able to image the atomic structure of materials if the atoms were far enough apart.[54] Around the turn of the century the electron optical components were coupled with computer control of the lenses and their alignment, enabling correction of aberrations. The first demonstration of aberration correction in TEM mode was by Harald Rose and Maximilian Haider in 1998 using a hexapole corrector, and in STEM mode by Ondrej Krivanek and Niklas Dellby in 1999 using a quadrupole/octupole corrector.[55]

As of 2025 correction of geometric aberrations is standard in many commercial electron microscopes, and they are extensively used in many different areas of science.[56][57] Similar correctors have also been used at much lower energies for LEEM instruments.[58]

Sample preparation

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An insect coated in gold for viewing with a scanning electron microscope (SEM)
Main articles: TEM Sample preparation, Ultramicrotomy, Staining, Cryofixation, Chemical milling, and Sputtering

Samples for electron microscopes mostly cannot be observed directly. The samples need to be prepared to stabilize the sample and enhance contrast. Preparation techniques differ vastly in respect to the sample and its specific qualities to be observed as well as the specific microscope used. Details can be found in the relevant main articles listed above.

Disadvantages

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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 and anti vibration mounts.[59]

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[60] 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 also been developed.[61]

Pleolipoviral virion (HRPV-6)[62]

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.[63][64][65]

Many samples suffer from radiation damage which can change internal structures. This can be due to either or both radiolytic processes or ballistic, for instance with collision cascades.[66] This can be a severe issue for biological samples,[67][68]

See also

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References

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[edit]
Revisions and contributorsEdit on WikipediaRead on Wikipedia

Electron microscope

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An electron microscope is a type of that uses a beam of accelerated electrons as a source of illumination to produce highly magnified images of specimens, achieving resolutions as fine as 0.1 nanometers, far surpassing the 200-nanometer limit of traditional microscopes due to the much shorter of electrons compared to visible . The instrument operates in a , where electrons are generated from a tungsten filament, accelerated by high voltages typically ranging from 20 to 120 kilovolts, and focused using electromagnetic lenses to form an image either by transmission through ultrathin samples or by scanning surfaces. There are two main variants: the transmission electron microscope (TEM), which passes electrons through a specimen less than 0.5 micrometers thick to reveal internal atomic structures at magnifications exceeding 1 million times, and the scanning electron microscope (SEM), which raster-scans a focused electron beam over a sample's surface to detect secondary or backscattered electrons, providing three-dimensional topographic details at resolutions around 1 nanometer and magnifications up to 400,000 times. These principles enable detailed analysis of material composition, crystal structures, and biological ultrastructures that are impossible to resolve with optical methods. The invention of the electron microscope traces back to 1931, when Ernst Ruska and Max Knoll at the Technical University of Berlin constructed the first prototype TEM, building on Louis de Broglie's 1924 wave-particle duality theory for electrons; Ruska later refined it into a practical instrument by 1933 and received the Nobel Prize in Physics in 1986 for this breakthrough. SEM development followed, with initial concepts proposed by Knoll in 1935 and a functional prototype achieved by Manfred von Ardenne in 1937. The first commercial TEM, the Siemens EM100, was introduced in 1939, marking the transition from laboratory curiosity to essential tool in scientific research. Since its inception, electron microscopy has transformed numerous disciplines by providing unparalleled insights into nanoscale phenomena, including viral structures in , defect in semiconductors for , and atomic arrangements in for applications. Modern advancements, such as aberration-corrected lenses and field-emission electron sources, have pushed resolutions below 0.5 angstroms, enabling even finer chemical and elemental mapping through techniques like . Despite challenges like requirements and operation, its superior resolving power continues to drive discoveries across the physical and life sciences.

Principles of operation

Electron optics and wave nature

The wave nature of electrons, first proposed by in 1924, enables their use in by associating a with particles based on their momentum. The de Broglie λ\lambda is given by the formula λ=h/p\lambda = h / p, where hh is Planck's constant and pp is the electron's momentum. For accelerated electrons, this wavelength is inversely proportional to their velocity, allowing high-energy electrons (typically 100–300 keV in microscopes) to achieve de Broglie wavelengths on the order of picometers, such as approximately 3.88 pm at 100 keV. This is orders of magnitude shorter than visible light wavelengths (around 400–700 nm), which limits light resolution to about 200 nm, whereas electrons enable sub-angstrom (less than 0.1 nm) imaging potential. In electron microscopy, the interaction of the electron beam with matter primarily involves scattering events that form the basis for image contrast. Elastic scattering occurs when an electron is deflected without significant energy loss, preserving its while changing direction, often through interactions with atomic nuclei; high-angle elastic events are described by Rutherford scattering, where the differential cross-section is proportional to 1/sin⁡4(θ/2)1/\sin^4(\theta/2) and θ\theta is the scattering . In contrast, inelastic scattering involves energy transfer to the sample, such as exciting inner-shell electrons or phonons, leading to energy losses of a few electron volts to thousands, which can generate signals like X-rays or but also broadens the beam. These interactions, dominated by elastic scattering for imaging due to its role in phase shifts and , allow electrons to probe atomic-scale structures far beyond light-based methods. Electron optics relies on to manipulate the beam, analogous to glass lenses in light microscopy but exploiting the charged nature of . The focusing principle stems from the , F=−e(v×B)\mathbf{F} = -e (\mathbf{v} \times \mathbf{B}), where ee is the electron charge, v\mathbf{v} is its velocity, and B\mathbf{B} is the ; in a or , the azimuthal field component causes off-axis to spiral inward, converging the beam at a focal point. Electromagnetic lenses, combining static with variable currents, enable adjustable focal lengths and beam control, forming images through successive focusing stages. However, imperfections in these fields introduce aberrations that degrade image quality. Spherical aberration in arises from the lens's inability to focus rays parallel to the optic axis at the same point, with peripheral rays focusing closer than paraxial ones due to stronger field gradients at larger radii, resulting in a blurred disk of confusion proportional to the third power of the aperture angle. occurs because with slightly different energies (from beam energy spread or ) experience varying refractive indices in the , causing lower-energy to focus shorter than higher-energy ones and further limiting resolution. These aberrations, inherent to static round lenses as proven by Scherzer's theorem in 1936, restrict practical resolutions despite the short de Broglie , though they can be mitigated in by optimizing lens design without altering the fundamental wave properties.

Resolution limits and magnification

The resolution in transmission electron microscopy (TEM) is fundamentally governed by the Abbe diffraction limit, adapted for as d=0.61λ/sin⁡αd = 0.61 \lambda / \sin \alpha, where dd is the minimum resolvable distance, λ\lambda is the de Broglie wavelength of the electrons, and α\alpha is the semi-angle of the objective . This formula highlights how shorter electron wavelengths—on the order of picometers at typical accelerating voltages—combined with optimized angles, enable resolutions far superior to light microscopy. However, practical implementation requires careful control of optical parameters to approach this theoretical bound. Magnification in electron microscopes is defined as the ratio of the size to the object size, M=M = size / object size, and serves to enlarge features for visualization. In TEM, practical magnification limits reach up to 50 million times, allowing detailed examination of atomic-scale structures, though higher values are often limited by specimen stability and detector capabilities rather than optical constraints. Several factors impose practical limits on resolution beyond the diffraction barrier, including lens aberrations such as spherical and chromatic effects, which distort the electron beam focus; beam divergence, which reduces coherence; and stability requirements for voltage, current, and mechanical vibrations that prevent drift during imaging. These elements collectively degrade image contrast and sharpness, necessitating precise instrumentation to mitigate their impact. A key distinction exists between point resolution and the information limit in electron microscopy. Point resolution refers to the smallest distance at which two points can be distinguished under ideal coherent illumination, primarily limited by in uncorrected systems. In contrast, the information limit accounts for partial coherence effects from and energy spread in the electron source, which dampen high-frequency information transfer through an envelope function, effectively reducing resolvable details. Modern aberration-corrected instruments achieve effective resolutions around 0.05 nm by pushing this information limit, enabling atomic-scale imaging in applications. Across electron microscope types, resolution varies significantly: TEM and scanning transmission electron microscopy (STEM) routinely attain sub-nanometer levels, often below 0.1 nm with corrections, while scanning electron microscopy (SEM) is typically limited to approximately 1 nm due to its surface-scanning nature and larger probe sizes.

History

Early development and invention

The discovery of the electron by J. J. Thomson in 1897 marked a pivotal moment in physics, as his experiments with cathode rays demonstrated that these rays consisted of negatively charged particles far smaller and lighter than atoms, which he termed "corpuscles" (later renamed electrons). This identification of the electron as a discrete particle provided the essential building block for subsequent technologies involving electron beams. Building on this, Louis de Broglie proposed in 1924 that all matter, including electrons, exhibits wave-particle duality, hypothesizing that particles possess an associated wavelength inversely proportional to their momentum. This revolutionary idea extended quantum concepts from light to matter, suggesting that electrons could be manipulated like waves for imaging purposes. The wave nature of electrons was experimentally verified in 1927 through the Davisson-Germer experiment, conducted at Bell Laboratories, where electrons diffracted off a lattice produced interference patterns consistent with de Broglie's predicted of approximately 0.165 nm for 54 eV electrons. This confirmation established electrons as suitable for high-resolution , far surpassing the wavelength limitations of visible (around 500 nm). These foundational concepts—electrons as particles with wave properties—directly inspired the development of electron microscopy in the late 1920s and early 1930s. In 1931, and Max Knoll at the constructed the first prototype transmission electron microscope, using magnetic coils as lenses to focus an electron beam and achieve a of 400× on a test grid, exceeding the resolution of contemporary light microscopes. This device demonstrated the feasibility of electron-optical but suffered from and low contrast. Independently, in May 1931, Reinhold Rüdenberg at Siemens-Schuckertwerke filed a for an electron microscope design using electrostatic lenses (German patent application, leading to in 1936). By 1933, Ruska refined the prototype with improved magnetic lenses, attaining a of 12,000× and enabling clearer of fine structures, such as etched sheets. A key innovation came in 1932 when Ruska, in collaboration with Bodo von Borries, patented a short-focal-length magnetic polepiece lens (German No. 680284), which minimized aberrations and allowed precise beam control essential for practical . This design became the basis for all subsequent magnetic microscopes. These advancements culminated in 1939 with producing the first commercial transmission electron microscope, the Super Microscope, capable of 20,000× magnification and resolutions down to 10 nm, marking the transition from laboratory prototypes to industrial tools. Parallel efforts in the during the 1930s, including early research at institutions like MIT, yielded prototypes such as the 1938 transmission electron microscope built at the (the first successful one in ), but these initial designs achieved only limited resolution and stability compared to German innovations.

Key advancements and commercialization

During , the emerged as a leader in development, driven by the need for advanced materials analysis in military applications. Vladimir Zworykin, leading a team at the Radio Corporation of America (RCA), oversaw the creation of improved transmission electron microscopes (TEMs) in the early 1940s, including a more affordable model demonstrated in 1942 that enabled broader wartime deployment for studying alloys and other materials. The invention of the (SEM) built on early prototypes from the 1930s. In 1935, Max Knoll constructed the first SEM prototype, demonstrating basic scanning principles for surface imaging. Two years later, in 1937, advanced the design with a scanning transmission system using a finely focused electron probe, laying foundational concepts for high-resolution surface examination. Commercialization followed postwar, with practical prototypes developed in the early 1950s by Charles Oatley and his team at the , culminating in the first commercial SEM, the Stereoscan, released by the Cambridge Scientific Instrument Company in 1965, which facilitated industrial adoption for materials characterization. Further innovations in the expanded electron microscopy capabilities. In 1966, Albert Crewe at the developed the scanning transmission electron microscope (STEM) with an annular dark-field detector, enabling atomic-scale imaging by collecting scattered electrons from a focused probe, which improved contrast for heavy atoms in biological and materials samples. Commercialization accelerated in the mid-20th century, particularly through Japanese manufacturers. began producing electron microscopes in the 1940s and entered the international market in the with reliable TEM models, while Electron Optics Laboratory (), founded in 1949, started manufacturing magnetic TEMs in 1950 and expanded globally by the mid-, making high-vacuum instruments more accessible for research and industry. By the 1970s, advancements in lens design and electron sources pushed TEM resolution to 0.2 nm, as exemplified by 's JEM-100B model introduced in 1968, which supported detailed crystallographic studies. The field's impact was recognized in 1986 when received the for his foundational work in and the design of the first electron microscope, highlighting its transformative role in scientific up to that point.

Types of electron microscopes

Transmission electron microscope (TEM)

The transmission electron microscope (TEM) is configured such that a beam of accelerated electrons passes through an ultra-thin specimen, generally less than 100 nm in thickness, to produce an image from the electrons transmitted through the sample. This electron-transparent preparation allows for the visualization of internal structures at the nanoscale, with the beam interacting via and absorption to generate contrast. The setup relies on electromagnetic lenses to focus the beam, similar to general principles. In TEM imaging, two primary modes are employed: bright-field and dark-field. Bright-field imaging collects the direct, unscattered transmitted electrons, resulting in areas of higher mass-thickness appearing darker due to increased scattering. Conversely, dark-field imaging utilizes scattered electrons, often those diffracted at specific angles, to highlight crystalline features and defects by blocking the direct beam. TEM finds extensive applications in revealing biological ultrastructures, such as the morphology of viruses including polyomaviruses and coronaviruses, enabling direct visualization and in clinical samples. In , it is used to examine defects like dislocations in metallic nanostructures and carbon-based materials, providing insights into phase evolution and atomic arrangements. These capabilities support fields from to nanomaterial engineering. TEM instruments achieve magnifications ranging from 10× to 1,000,000×, with resolutions approaching 0.1 nm in aberration-corrected systems, allowing atomic-scale . Historically, the first practical applications of TEM in occurred in the , when Robert Heidenreich at used electrolytic thinning to study metal textures in 1949.

Scanning electron microscope (SEM)

The scanning electron microscope (SEM) operates by directing a finely focused beam of electrons across the surface of a sample in a raster-scanning pattern, point by point, to generate images based on the emitted signals from electron-sample interactions. This sequential scanning allows for the construction of detailed topographic maps of the specimen's surface, with the intensity of detected signals determining the brightness of each pixel in the resulting image. SEM accommodates bulk samples up to several centimeters in scale, unlike transmission-based methods that require ultrathin sections, enabling the examination of three-dimensional objects without extensive dissection. For biological samples, standard SEM requires chemical fixation to preserve structure (which kills the cells), followed by dehydration, critical point drying, and coating with a conductive material such as gold or carbon to mitigate charging effects in the high vacuum environment. Additionally, environmental SEM variants operate at lower levels, facilitating of hydrated or uncoated specimens such as biological materials—including wet cells or bacteria—allowing observation of dynamic surface processes without drying or coating, though long-term cell viability may be limited by the reduced vacuum and electron beam exposure. The SEM's is approximately 300 times greater than that of optical microscopes, owing to the small interaction volume of the beam with the sample surface, which allows sharp focus across a wide range of heights in the specimen. Typical SEM resolution ranges from 0.5 to 10 nm, depending on beam energy, probe current, and , while magnification can reach up to 1,000,000× for high-detail surface features. These capabilities arise from the short de Broglie of electrons and precise beam control, enabling visualization of nanoscale surface structures. Emitted signals, such as for and backscattered electrons for compositional contrast, are detected to form the image. SEM finds widespread applications in analyzing surfaces to identify failure mechanisms in materials, such as crack propagation paths in metals or composites. In , it reveals intricate surface details on specimens like exoskeletons or cellular membranes after appropriate coating. Forensic investigations employ SEM to examine , including particles or fiber morphologies for evidentiary matching.

Scanning transmission electron microscope (STEM)

The scanning transmission electron microscope (STEM) is a versatile instrument that integrates the scanning probe approach with transmission electron microscopy principles, enabling high-resolution imaging of thin specimens at the atomic scale. In STEM, a finely focused beam is raster-scanned across the sample, allowing for the collection of transmitted electrons to form images that reveal internal and composition with exceptional detail. This hybrid facilitates simultaneous acquisition of multiple signal types, making it particularly suited for analytical studies in . The basic setup of a STEM involves a high-brightness electron source, such as a , which generates a probe beam converged to a sub-angstrom and scanned over the specimen using deflection coils. The thin sample, typically less than 100 nm thick, is positioned such that electrons transmit through it, interacting via elastic and processes. Multiple detectors are arranged below the sample to capture these transmitted electrons at various angles: a bright-field detector collects unscattered or low-angle scattered beams for phase-contrast information, while segmented or annular detectors record higher-angle for amplitude-contrast details. This configuration allows for real-time imaging and simultaneous from diverse signals, enhancing the efficiency of nanoscale . The development of STEM traces back to the pioneering efforts of Albert Crewe and his team at the in the late 1960s and 1970s, who constructed the first practical instrument capable of atomic-resolution imaging using a field-emission source and annular detectors. Crewe's innovations, including the demonstration of single-atom visibility in 1970, laid the foundation for modern STEM by emphasizing Z-contrast imaging over conventional bright-field modes. By the 1980s, commercial STEM systems emerged, and with the advent of aberration correction in the 2000s, STEM became a standard tool in analytical electron microscopy for its ability to combine high spatial resolution with spectroscopic capabilities. Today, STEM instruments are integral to advanced laboratories worldwide, supporting multidisciplinary research from to . A key imaging mode in STEM is annular dark-field (ADF) imaging, which employs a ring-shaped detector to collect high-angle scattered electrons, producing Z-contrast images where intensity scales with the square of the (Z²) due to incoherent Rutherford scattering. This sensitivity to atomic number enables clear visualization of heavy elements against lighter backgrounds without the phase contrast artifacts common in bright-field TEM, making ADF-STEM ideal for locating impurities or mapping elemental distributions in complex alloys. The contrast arises primarily from thermal diffuse scattering at elevated angles, minimizing contributions from crystalline diffraction and providing chemically intuitive images. STEM finds extensive applications in atomic-scale materials analysis, particularly for studying catalyst nanoparticles where it reveals distributions and structural dynamics under operational conditions. For instance, STEM observations of platinum-based catalysts during reactions provide insights into particle and facet evolution, aiding the design of more efficient heterogeneous catalysts. Another prominent application is 4D-STEM, which records a full pattern at each scan position to enable strain mapping, quantifying lattice distortions in semiconductors or 2D materials with nanometer precision. This technique has been instrumental in analyzing strain fields around defects in heterostructures, informing device performance optimization. With aberration correction, STEM achieves sub-0.05 nm resolution, allowing direct imaging of atomic columns and even light elements like oxygen in oxides. This breakthrough, realized in instruments like the TEAM 0.5 project microscope in , extends the probe size limit from ~0.1 nm to below 50 pm, enabling quantitative analysis of beam-sensitive materials without significant damage. Such resolutions have revolutionized fields like research by permitting the observation of subtle bonding arrangements and electronic inhomogeneities.

Instrumentation and components

Electron sources

Electron sources in electron microscopes generate beams of electrons that are accelerated and focused to illuminate specimens, with the source type determining key beam properties such as , coherence, and stability. The primary types include guns and field emission guns (FEGs), each suited to different demands based on their emission mechanisms and performance characteristics. Thermionic guns, the most common for standard applications, rely on heating a filament to emit electrons via thermal excitation. Typically, these use a hairpin filament heated to approximately 2700 K, producing a beam with moderate brightness of about 10^5 A/cm² sr. This design is robust and cost-effective, making it widely adopted in conventional transmission electron microscopes (TEMs) and scanning electron microscopes (SEMs) where ultra-high resolution is not required. However, the larger source size (crossover diameter around 50 μm) limits beam coherence compared to advanced sources. Field emission guns (FEGs) offer superior performance for high-resolution imaging by extracting electrons through quantum tunneling under a strong applied to a sharply pointed . These include cold field emission guns, operating at , and Schottky (thermal-assisted) FEGs, which heat the emitter slightly to enhance stability. FEGs achieve much higher brightness, typically 10^8 to 10^9 A/cm² sr, enabling smaller probe sizes and better signal-to-noise ratios in demanding applications. Their use is prevalent in advanced and SEMs for atomic-scale imaging. A fundamental limitation of all electron sources is , arising from the Poisson statistics of electron emission, where the variance in electron count equals the mean number of electrons, leading to fluctuations that degrade quality. Additionally, the energy spread (ΔE) of the emitted electrons—typically 1–3 eV for thermionic guns and 0.3–0.7 eV for FEGs—contributes to downstream, as electrons of varying energies focus differently. This spread is narrower in FEGs due to the absence of broadening, improving resolution in aberration-sensitive systems. Electrons from the source are accelerated by voltages ranging from 1 kV in low-energy SEMs to 400 kV in high-voltage TEMs, with 100–300 kV often providing an optimal balance between achieving short de Broglie wavelengths for high resolution and minimizing beam-induced sample in sensitive materials. Higher voltages reduce the relative impact of spread on aberrations but increase penetration and potential structural alteration in specimens.

Lenses and beam control

In electron microscopes, electromagnetic lenses serve as the primary optical elements for focusing and directing the beam, replacing glass lenses used in light microscopy due to the inability of materials to refract electrons effectively. These lenses operate on the principle of the , where a generated by current-carrying coils deflects charged electrons toward the . designs consist of helical windings around a soft iron core to produce a uniform axial , while aperture designs incorporate pole pieces and a central to shape the field more precisely, enhancing focusing power and reducing aberrations. The ff of such a is approximated by f≈VeB2r2/(8m),f \approx \frac{V}{e B^2 r^2 / (8 m)}, where VV is the accelerating voltage, ee is the charge, BB is the strength, rr is the bore , and mm is the rest ; this relation highlights the lens's sensitivity to field strength and beam energy. Condenser lenses, typically one or two in series above the sample, control beam convergence and illumination intensity by adjusting the current to vary their , allowing the beam—emanating from the source—to be demagnified into a fine probe or diverged for parallel illumination in experiments. The objective lens, positioned closest to the sample, collects and focuses transmitted or scattered electrons to form the initial image, with its short (often 1–5 mm) enabling high up to 1 million times while determining key resolution limits through beam-sample interactions. Together, these lenses form a compound optical system that achieves sub-angstrom precision in modern instruments. In scanning modes, such as those employed in scanning electron microscopes (SEM) and scanning transmission electron microscopes (STEM), scanning coils provide beam deflection via orthogonal magnetic fields, rastering the focused probe across the sample in a pixel-by-pixel pattern to build images sequentially. These coils enable scan speeds up to 10610^6 points per second, balancing acquisition time with signal quality for dynamic or large-area imaging. Alignment systems, including stigmators and deflectors, ensure beam symmetry; stigmators apply differential currents to correct arising from lens imperfections, while deflectors fine-tune the beam path to center it on the , preventing off-axis aberrations and maintaining resolution.

Detectors and vacuum systems

In electron microscopes, detectors are crucial for capturing the various signals generated by electron-sample interactions, such as transmitted electrons in (TEM) and in scanning electron microscopy (SEM). For transmitted electrons, which pass through thin samples to form images based on beam intensity, detectors typically consist of a that converts energy into , coupled to a or for signal amplification and detection. This setup enables high-sensitivity imaging of internal sample structures by quantifying the transmitted beam's variations. In contrast, the Everhart-Thornley detector, widely used for —low-energy electrons emitted from the sample surface providing topographic information—employs a biased to attract and accelerate these electrons, followed by a for efficient signal collection. Electron microscopes require high vacuum environments to minimize electron scattering by residual gas molecules, ensuring a mean free path sufficient for the electron beam to reach the sample unaltered. Operating pressures typically range from ~1 Pa (or higher, up to ~1000 Pa) in low-vacuum or environmental scanning modes to 10^{-10} Pa in transmission systems, with conventional high-vacuum SEM and TEM operating around 10^{-5} to 10^{-7} Pa. To achieve these levels, turbomolecular pumps, which use high-speed rotating blades to transfer momentum to gas molecules, are employed for initial high-vacuum evacuation, often backed by rotary vane roughing pumps. pumps, utilizing strong to ionize and bury gas molecules into a cathode, maintain ultra-high vacuums without moving parts, complementing turbomolecular systems in critical regions like the . Differential pumping is essential in electron microscopes to accommodate varying pressure requirements across components, preventing contamination while allowing sample introduction. This involves separate vacuum chambers connected by small apertures: the electron gun chamber is maintained at approximately 10^{-7} Pa to protect the source from outgassing, while the sample chamber in SEMs operates at around 10^{-5} Pa for standard imaging. Turbomolecular and ion pumps are strategically placed in each stage to sustain these gradients, with the aperture limiting gas flow between compartments. For imaging hydrated or beam-sensitive samples, variable pressure SEM (VP-SEM) or environmental SEM (ESEM) adaptations introduce controlled gas pressures up to several hundred Pa in the sample chamber, using differential pumping to isolate the high-vacuum column. This allows observation of wet biological specimens without dehydration, as water vapor neutralizes charging and maintains sample integrity during secondary electron imaging.

Imaging and analytical modes

Surface imaging modes

Surface imaging modes in scanning electron microscopy (SEM) primarily rely on electrons emitted from the sample surface following interaction with the incident electron beam. These modes utilize (SE) and backscattered electrons (BSE) to generate images that reveal and compositional variations, respectively. By scanning the focused beam across the sample and detecting these emitted signals, high-resolution surface details can be visualized without requiring transmission through the specimen. Secondary electrons are low-energy electrons, typically with energies less than 50 eV, generated through events where the incident beam excites and ejects electrons from the sample's outer atomic shells. These SEs originate from a very shallow depth, with an escape depth of approximately 1-5 nm, making them highly sensitive to surface and providing excellent contrast for features like edges, roughness, and texture. In SE imaging, the yield of detected electrons varies with local surface orientation relative to the detector, enhancing the three-dimensional appearance of the image. Backscattered electrons, in contrast, are high-energy electrons (generally >50 eV, approaching the incident beam energy) produced by within the sample, where primary electrons are redirected back toward the surface. BSEs emerge from a larger interaction , on the order of micrometers in depth and lateral extent, and their yield depends strongly on the (Z) of the elements present, resulting in Z-contrast imaging where heavier elements appear brighter due to higher backscattering efficiency. This mode is particularly useful for distinguishing phases or inclusions in materials based on composition, though it offers less topographic detail than SE imaging. The efficiency of SE and BSE emission is characterized by yield curves, which plot the secondary electron yield δ(E₀) and backscattered electron yield η(E₀) as functions of the incident beam energy E₀. The SE yield δ(E₀) typically rises from near zero at low E₀, peaks at a maximum (often around 200-1000 eV depending on the material), and then decreases asymptotically, reflecting the balance between electron generation and escape probability. Similarly, the BSE yield η(E₀) increases monotonically with E₀ and atomic number, approaching values up to 0.5-0.6 for high-Z materials at keV energies, which informs optimal beam conditions for imaging. These curves, derived from experimental measurements and Monte Carlo simulations, guide the selection of accelerating voltage to maximize signal while minimizing beam damage or charging. For non-conductive samples, which can accumulate charge under the electron beam and distort images, variable pressure (or environmental) SEM modes introduce a low-pressure gas (typically 0.1-10 of or ) into the specimen chamber to facilitate charge neutralization. The incident electrons ionize the gas molecules, producing positive ions that are attracted to negatively charged regions on the sample surface, while free electrons from can also contribute to balancing the charge; this gas-mediated process allows direct of insulators like biological tissues or polymers without conductive coatings. The presence of gas slightly scatters the primary beam and amplifies secondary signals through cascades, but it enables hydrated or wet sample observation under near-native conditions.

Transmission and diffraction modes

In transmission electron microscopy (TEM), transmitted electrons are utilized to reveal internal structures and crystallographic details of thin specimens. These modes exploit the interaction of the electron beam with the sample, where unscattered or minimally scattered electrons pass through, while diffracted electrons provide information on atomic arrangements. Key techniques include bright-field imaging, dark-field imaging, electron diffraction patterns, and phase contrast methods, each offering distinct contrast mechanisms for analyzing materials from metals to biomolecules. Bright-field TEM imaging employs the direct, unscattered electron beam to form the image, with an objective aperture blocking scattered electrons to enhance contrast. In this mode, regions of the sample that transmit more electrons appear brighter, while areas with higher mass density or thickness scatter electrons more effectively, resulting in darker features due to mass-thickness contrast. This contrast arises because heavier atoms or thicker sections increase electron scattering probability, reducing the intensity of transmitted electrons reaching the detector. Bright-field mode is particularly effective for visualizing overall morphology and identifying dense inclusions in materials like alloys or biological tissues stained for enhanced scattering. Dark-field TEM imaging, in contrast, selects diffracted electrons by centering a specific diffraction spot on the optic axis while blocking the direct beam, producing a dark background with bright features from scattering sites. This mode highlights crystal defects such as dislocations or stacking faults, as these regions bend the lattice and direct diffracted electrons into the aperture, increasing local intensity. It is also valuable for distinguishing crystallographic phases in polycrystalline samples by selectively imaging electrons from particular orientations. By emphasizing scattered electrons, dark-field reveals structural heterogeneities that may be obscured in bright-field views. Electron diffraction modes provide crystallographic information by analyzing the angular distribution of transmitted electrons. In selected area electron diffraction (SAD), a small aperture selects a region (typically 0.5–5 μm) of the sample, and a parallel electron beam produces a spot pattern on the detector, where spot positions correspond to reciprocal lattice vectors. These patterns obey Bragg's law, given by nλ=2dsin⁡θn\lambda = 2d \sin\theta where nn is an integer, λ\lambda is the electron wavelength, dd is the interplanar spacing, and θ\theta is the diffraction angle; measuring θ\theta allows determination of dd for phase identification and orientation mapping. Convergent beam electron diffraction (CBED) improves resolution by using a focused, convergent beam (probe size <50 nm) to generate disk-like patterns with internal symmetry, enabling point group determination and thickness measurement from higher-order Laue zones. CBED offers higher spatial precision than SAD for local structure analysis in nanomaterials. Phase contrast modes enhance visibility of weakly scattering objects, such as unstained biomolecules, by converting phase shifts in the electron wave to amplitude differences. The defocus method achieves this by slightly underfocusing the objective lens, transferring low-spatial-frequency phase information into contrast, though it limits resolution due to spherical aberration at larger defocus values. The Zernike phase plate method inserts a thin-film plate (e.g., amorphous carbon, ~30 nm thick with a central hole) in the back focal plane to shift the phase of unscattered electrons by π/2\pi/2 relative to scattered ones, providing uniform contrast at focus without resolution loss. This approach has enabled high-resolution imaging of proteins like at ~1.2 nm and viruses, improving signal-to-noise for cryo-TEM applications.

Spectroscopic analysis modes

Spectroscopic analysis modes in electron microscopy utilize the interactions between the electron beam and sample to extract chemical composition, electronic structure, and bonding information. These techniques rely on detecting emitted or energy losses from inelastic scattering events, enabling elemental identification and quantification at high spatial resolutions. In (TEM) and (STEM), such modes complement imaging by providing analytical data from regions as small as a few nanometers. Energy-dispersive X-ray spectroscopy (EDS), also known as EDX, detects X-rays generated when incident electrons interact with sample atoms, producing both a continuous bremsstrahlung spectrum from decelerated electrons and characteristic X-rays unique to each element's atomic structure. The bremsstrahlung background provides information on beam energy, while characteristic peaks allow elemental identification from beryllium to uranium, with detection limits around 0.1 wt% for most elements. Quantification in thin specimens, typically less than 100 nm thick, employs the Cliff-Lorimer method, which relates the ratio of characteristic X-ray intensities from two elements to their concentration ratio using experimentally determined k-factors, assuming negligible absorption and fluorescence effects. This approach, developed for STEM applications, achieves accuracies of 1-5% relative error for major elements in alloys and minerals. Electron energy-loss spectroscopy (EELS) measures the energy lost by transmitted electrons due to inelastic scattering, typically in the range of 10-1000 eV, revealing details about electronic excitations and core-level transitions. Low-loss EELS (<50 eV) probes plasmons and valence electrons, while core-loss EELS (>50 eV) identifies elements via ionization edges analogous to X-ray absorption. The fine structure near these edges, known as energy-loss near-edge structure (ELNES), encodes bonding and coordination information by reflecting the density of unoccupied states; for example, shifts in the silicon L-edge ELNES indicate Si-O versus Si-Si bonding environments. EELS offers superior energy resolution (0.1-1 eV) compared to EDS, enabling analysis of light elements like lithium and hydrogen, though it requires thinner samples (<50 nm) to minimize multiple scattering. Wavelength-dispersive spectroscopy (WDS) provides higher energy resolution (about 5-20 eV) than EDS (typically 130 eV), making it particularly effective for resolving overlapping peaks and quantifying light elements such as boron, carbon, nitrogen, and oxygen in electron microprobes and TEM attachments. By diffracting X-rays with a curved crystal analyzer tuned to specific wavelengths via Bragg's law, WDS achieves lower detection limits (down to 10 ppm) and better peak-to-background ratios, though it requires sequential scanning and longer acquisition times. This mode is valuable for precise compositional analysis in geological and materials samples where EDS sensitivity to light elements is limited. In STEM, spectrum imaging combines raster scanning with EDS or EELS acquisition at each pixel to generate nanoscale elemental maps, achieving spatial resolutions of 1-5 nm for heavy elements and 5-10 nm for lighter ones. This technique produces hyperspectral datasets where elemental distributions are extracted via background subtraction and peak fitting, enabling visualization of segregation, precipitates, or diffusion profiles; for instance, in bimetallic nanoparticles, it reveals core-shell structures with atomic percent precision. Such mapping supports correlative studies of structure-property relationships in nanomaterials.

Advanced techniques and instruments

Aberration correction

Aberration correction in electron microscopes addresses the inherent imperfections of electromagnetic lenses, primarily spherical and chromatic aberrations, which limit spatial resolution by blurring the electron probe. Spherical aberration, characterized by the coefficient CsC_s, causes electrons at different radial distances from the optical axis to focus at varying points, degrading image sharpness; this can be corrected using multipole lens systems, such as quadrupole-octupole configurations that dynamically adjust the electron trajectories to compensate for these deviations. The quadrupole-octupole corrector, developed by Krivanek and colleagues, was first demonstrated in 1999 on a scanning transmission electron microscope (STEM), enabling sub-angstrom probe sizes by expanding the usable angular aperture without introducing additional aberrations. Chromatic aberration arises from variations in electron energy, leading to defocusing of off-energy electrons; correction methods include energy filtering to select a narrow energy bandwidth or Wien filters, which employ crossed electric and magnetic fields to counteract energy-dependent deflections without dispersing the beam spatially. Wien filters, as implemented in modern correctors, provide precise chromatic correction (CcC_c) by operating in a velocity-mapping mode that aligns trajectories for electrons of different energies. These systems, often integrated with spherical aberration correctors, have been refined since the early 2000s to handle both axial and off-axial aberrations comprehensively. The implementation of aberration correction has dramatically enhanced resolution in STEM, reducing the information limit from approximately 0.2 nm to below 0.05 nm at typical accelerating voltages of 100–300 kV, as achieved in early corrected instruments like the TEAM 0.5 microscope. This improvement facilitates atomic-resolution annular dark-field (ADF) STEM imaging, where high-angle scattering provides Z-contrast sensitive to atomic number, allowing visualization of light elements such as oxygen and carbon alongside heavier atoms in complex materials. By minimizing probe tails and enabling brighter, smaller probes, correction suppresses background noise, improving signal-to-noise ratios for quantitative analysis of atomic structures and defects. Commercial adoption began in the early 2000s with systems from Nion, Inc., featuring proprietary quadrupole-octupole correctors for STEM, and CEOS GmbH, offering hexapole-based correctors adaptable to transmission electron microscopes (TEM) and STEM platforms. These technologies, initially prototypes, became standard in high-end instruments by the 2010s, with over 500 corrected microscopes installed globally, enabling routine sub-0.1 nm imaging in materials science and nanotechnology research. Ongoing advancements, such as integrated fifth-order corrections, continue to push resolution limits while maintaining stability for long-duration experiments.

Cryo-electron microscopy

Cryo-electron microscopy (cryo-EM) is a specialized imaging technique that enables the visualization of biological specimens in their near-native, frozen-hydrated state, preserving delicate structures without the artifacts introduced by chemical fixation or staining. Although the rapid freezing process renders cells effectively dead, it maintains structural integrity close to the living state. By rapidly freezing samples to form vitreous ice, cryo-EM minimizes dehydration and structural distortion, allowing high-resolution imaging of macromolecules and cellular components at cryogenic temperatures. This method builds on transmission electron microscopy (TEM) principles but incorporates low-temperature environments to reduce beam-induced damage and maintain sample integrity. The vitrification process is central to cryo-EM, involving the ultra-rapid freezing of aqueous biological samples to prevent ice crystal formation, which would otherwise damage cellular structures. Samples are typically applied to a grid, blotted to thin the suspension, and plunge-frozen into liquid ethane cooled by liquid nitrogen, achieving cooling rates exceeding 10^5 K/s to form amorphous (vitreous) ice. This non-crystalline state preserves the hydrated conformation of proteins and complexes, avoiding the disruptive effects of hexagonal or cubic ice crystals. Automated systems like the Vitrobot facilitate reproducible vitrification, ensuring uniform ice thickness for optimal imaging. In cryo-TEM and cryo-scanning TEM (cryo-STEM), specimens are imaged using specialized stages maintained at approximately 90-100 K to keep the vitreous ice stable and suppress thermal motion. The electron beam is operated at low doses, typically 10-50 electrons/Ų, to minimize radiation damage, which can cause bond breakage and conformational changes in sensitive biomolecules. Direct electron detectors and phase plates enhance signal-to-noise ratios under these conditions, enabling the capture of high-fidelity projections. Cryo-STEM complements cryo-TEM by providing annular dark-field imaging for thicker samples, further reducing dose requirements through focused probes. Single-particle analysis in cryo-EM involves collecting thousands to hundreds of thousands of two-dimensional projections of purified macromolecules in random orientations, followed by computational alignment and 3D reconstruction to achieve resolutions often better than 3 Å, typically in the range of 1.5-4 Å. This approach, pioneered by Joachim Frank, uses algorithms like RELION or cryoSPARC to classify particles, correct for heterogeneity, and generate density maps that reveal atomic details without averaging over crystalline arrays. For flexible proteins, advanced methods such as 3D variability analysis capture conformational dynamics from the dataset. Key advances in cryo-EM were recognized by the 2017 Nobel Prize in Chemistry, awarded to Jacques Dubochet for developing vitrification, Joachim Frank for single-particle reconstruction, and Richard Henderson for demonstrating atomic-resolution potential. Complementing these, cryo-electron tomography (cryo-ET) extends the technique to in situ cellular imaging, acquiring tilt series from frozen sections to reconstruct 3D tomograms of macromolecular arrangements within their native context, often at 3-10 Å resolution. By 2025, cryo-EM has become routine for protein structure determination, with over 30,000 entries in the Protein Data Bank, including landmark structures like the SARS-CoV-2 spike protein that informed vaccine design.

Sample preparation

Methods for biological samples

Biological samples for electron microscopy require careful preparation to preserve their delicate, hydrated structures while ensuring compatibility with the instrument's high vacuum environment. Conventional preparation for transmission electron microscopy (TEM) and scanning electron microscopy (SEM) typically requires chemical fixation, dehydration (or drying and coating for SEM), and often thin sectioning, processes that render cells non-viable due to incompatibility with vacuum conditions and structural demands. In contrast, cryo-TEM employs rapid freezing for vitrified preservation of hydrated, near-native structures without chemical fixatives, though freezing itself constitutes cell death; environmental SEM (ESEM) enables imaging of hydrated or short-term live samples at reduced pressures, avoiding drying. Fixation is the initial step, stabilizing cellular components against subsequent processing. Chemical fixation commonly employs glutaraldehyde, a cross-linking agent that penetrates tissues and reacts with proteins to form stable bonds, typically at concentrations of 2-4% in a buffered solution such as 0.1 M sodium cacodylate at pH 7.4 for 1-2 hours or overnight at 4°C. This is often followed by secondary fixation with osmium tetroxide (1-2%) to enhance membrane preservation by reacting with lipids, applied for 30-60 minutes at room temperature. Cryo-fixation alternatives, such as plunge-freezing into liquid ethane cooled by liquid nitrogen, rapidly vitrify samples (up to 10-20 μm thick) without ice crystal formation, preserving native hydration and ultrastructure in milliseconds. High-pressure freezing (HPF) extends this to thicker specimens (up to 200 μm) by applying 2,000 bar pressure during freezing to suppress ice nucleation, commonly using devices like the Leica EM HPM100. After fixation, samples are dehydrated using a graded series of ethanol (30-100%) to remove water, which is incompatible with epoxy resin embedding, followed by infiltration with resin such as Epon or LR White over 24-48 hours. Sectioning via ultramicrotomy produces thin slices suitable for transmission electron microscopy (TEM), typically 50-100 nm thick to minimize beam scattering while allowing electron transmission. This process uses an ultramicrotome equipped with a diamond knife, where the embedded block advances incrementally (e.g., 70 nm per section) against the knife edge at speeds of 0.5-2 mm/s, collecting ribbons of sections on copper grids floated on water in the knife boat. Diamond knives, with edge radii below 10 nm, provide superior durability and sharpness for biological resins compared to glass knives, reducing compression artifacts during cutting at room temperature or cryotemperatures (-90 to -110°C for frozen-hydrated samples). Contrast enhancement through is essential for visualizing cellular in TEM, as biological tissues inherently offer low . Heavy metal stains like uranyl (2-4% aqueous or methanolic) to proteins, nucleic acids, and , providing by electrons around structures; sections are immersed for 5-10 minutes followed by rinsing. Lead citrate (Reynolds' ) is then applied for 2-5 minutes to further stain proteins and ribonucleoprotein granules, often yielding double-contrast images where membranes appear as lines. For cryo-prepared samples, freeze-substitution replaces with organic solvents (e.g., acetone containing uranyl and ) at -90°C over 48-72 hours, gradually warming to before , to maintain structural fidelity without recrystallization. Despite these methods, challenges persist in preserving biological integrity. Dehydration during chemical processing can induce shrinkage artifacts, with tissues contracting up to 20-30% due to loss of hydration and lipid extraction, distorting relative dimensions and creating voids. Beam-induced damage in the microscope further exacerbates shrinkage, as hydrated or resin-embedded samples lose mass under electron irradiation, leading to bubbling or cracking; this is mitigated by low-dose imaging but remains a limitation for beam-sensitive organics. Cryo-methods reduce such artifacts by avoiding dehydration, though they require specialized equipment and may introduce devitrification if not handled below -130°C.

Methods for inorganic materials

Sample preparation for inorganic materials such as metals, ceramics, and semiconductors in electron microscopy focuses on achieving a clean, thin, and structurally representative surface or section to reveal microstructure without introducing artifacts. These rigid samples typically require mechanical and milling techniques to thin them to electron transparency (often <100 nm for TEM) or conductivity (for SEM), contrasting with softer biological specimens. Key methods include polishing, milling, coating, and etching, selected based on material hardness and analysis goals. Mechanical polishing is a foundational step for preparing bulk inorganic samples, particularly hard and brittle ones like ceramics and metals, to create a flat surface suitable for further thinning. The process begins with grinding using silicon carbide papers (from 400 to 1200 grit) to remove excess material, followed by sequential polishing with diamond abrasives on cloths or films, starting at 9 μm and progressing to <1 μm particle size for a mirror-like finish. This yields a scratch-free surface that minimizes topographic artifacts in SEM imaging or serves as a precursor for TEM foil preparation. For metals, the polished disc (typically 3 mm diameter) is then often electropolished to achieve electron transparency; in twin-jet electropolishing, the sample acts as the anode in an electrolyte bath (e.g., perchloric acid in acetic anhydride for aluminum), with controlled voltage (10-30 V) and temperature to dissolve material preferentially at high-current-density regions, producing foils ~50-100 nm thick at the perforation center. This method is widely used for metals like steel and aluminum alloys, ensuring damage-free thinning for high-resolution TEM. Focused ion beam (FIB) milling enables site-specific preparation of inorganic samples, ideal for semiconductors and layered ceramics where precise cross-sections are needed. A gallium (Ga+) ion beam, accelerated at 30 kV, is raster-scanned to sputter material, creating lamellae ~100 nm thick and 10-20 μm wide by protective platinum deposition followed by milling from both sides. This technique preserves local microstructure, such as interfaces in silicon wafers or nanoparticle composites, and is essential for 3D tomography in FIB-SEM workflows. Unlike broad mechanical methods, FIB allows targeted extraction from bulk samples without extensive handling. For non-conductive inorganic samples like ceramics or oxides in SEM, coating via sputtering enhances imaging by preventing charge buildup under the electron beam. A thin layer (~5-10 nm) of conductive material is deposited in a vacuum chamber using a magnetron sputter coater; gold (Au) provides high conductivity and fine grains (~2 nm) for topographic detail, while carbon (C) is preferred for energy-dispersive X-ray spectroscopy (EDS) to avoid spectral overlap. The process involves plasma ionization of argon gas to eject target atoms onto the sample, typically at 10-50 mA for 30-60 seconds, ensuring uniform coverage without altering the underlying structure. Etching techniques reveal fine microstructural features like grain boundaries in polished inorganic samples by selectively removing material. Chemical etching involves immersion in reagents such as alcoholic nitric acid for steels or oxalic acid for high-alloy metals, which preferentially attacks boundaries due to higher reactivity, creating topographic contrast visible in SEM (e.g., boundaries appear as dark valleys in secondary electron mode). Ion etching, using argon ions at 4-6 kV in a broad-beam miller, provides damage-free thinning and boundary delineation for ceramics, often at a 15-18° incidence angle to avoid redeposition. These methods enhance phase contrast without excessive material removal, typically etching for 10-30 seconds to 1-2 μm depth. These preparation methods support critical applications in characterizing inorganic microstructures, such as assessing nanoparticle dispersion in metal matrices via drop-casting diluted suspensions onto TEM grids followed by FIB lift-out for 3D analysis, or identifying phases in ceramics through etched SEM images combined with EDS mapping. For instance, electropolished foils enable diffraction patterns for phase confirmation in alloy precipitates, while coated and etched samples reveal grain boundary segregation in semiconductors, informing material performance in electronics and catalysis.

Limitations and challenges

Technical and operational drawbacks

Electron microscopes are notoriously expensive, with transmission electron microscopes (TEMs) typically costing from around $80,000 for entry-level models to exceeding $3 million for advanced systems as of 2025, while scanning electron microscopes (SEMs) range from $60,000 to $150,000 for benchtop models to over $1 million for high-resolution systems depending on features. Maintenance is equally intensive, involving annual service contracts that can exceed $100,000 for high-resolution systems as of 2025 due to the need for specialized parts, regular calibration, and vacuum system upkeep. A primary operational limitation stems from the high- environment required for beam , which scatters electrons in gaseous atmospheres and prevents of live or hydrated samples without specialized enclosures; though low-vacuum or environmental SEMs allow of hydrated specimens in variable modes. This constraint necessitates extensive sample preparation, often taking hours to days, including , fixation, and sectioning to ensure compatibility. Throughput in electron microscopy is inherently low compared to light microscopy, as imaging occurs serially—scanning point-by-point or through thin sections—rather than capturing parallel fields of view, resulting in longer acquisition times for large areas or volumes. Radiation damage further complicates operations, with knock-on displacement prevalent in inorganic materials under high-energy beams, causing atomic ejection above threshold energies around 200-300 keV, while dominates in organic specimens, leading to bond breakage and structural degradation at electron doses exceeding 20-50 electrons per square . Effective use of electron microscopes demands significant operator expertise, including precise alignment of electromagnetic lenses, optimization of beam parameters, and skilled interpretation of high-contrast images, often requiring years of specialized training equivalent to graduate-level proficiency in materials or biological applications.

Artifacts and sample damage

In electron microscopy, charging artifacts arise when non-conductive samples accumulate electric charge from the incident electron beam, leading to deflection of the beam and resulting image distortions such as bright or dark spots and geometric aberrations. This effect is particularly pronounced in scanning electron microscopy (SEM) of insulating materials, where the lack of conductivity prevents charge dissipation, causing the sample surface to act as a dynamic lens that shifts image focus. To mitigate charging, samples are often coated with a thin conductive layer, such as carbon or gold, which allows electrons to dissipate without significantly altering surface topography. Drift artifacts manifest as blurring or distortion in images due to unintended sample movement relative to the beam, often caused by thermal vibrations or mechanical instabilities in the microscope stage. Contamination, meanwhile, occurs from the buildup of hydrocarbons or other residues on the sample surface under the beam, forming a carbonaceous layer that further blurs features and reduces contrast over time. These issues are exacerbated in high-vacuum environments, where residual gases can polymerize under electron irradiation, leading to gradual image degradation during prolonged exposure. Beam damage encompasses several mechanisms induced by electron bombardment, including radiolytic bond breaking, knock-on displacement, and thermal effects. Heating results from inelastic scattering that converts beam energy into phonon excitations, with the temperature rise proportional to the beam current and inversely proportional to the sample's thermal conductivity and the size of the irradiated area; this can cause structural changes or melting in sensitive materials. Sputtering involves the ejection of atoms from the sample surface due to momentum transfer from high-energy electrons, leading to erosion and topographic alterations, while bubbling in polymers arises from radiolysis-induced gas evolution that forms voids and disrupts morphology. These damage types limit the achievable resolution, particularly for beam-sensitive specimens like biological tissues or nanomaterials. Contrast artifacts in transmission electron microscopy (TEM) include overfocus halos, which appear as bright Fresnel fringes around features in phase contrast due to defocus enhancing edge at the expense of accuracy. Thickness fringes, or interference patterns from electron wave interactions in wedge-shaped samples, can mimic structural details and mislead interpretation of material density variations. Mitigation strategies for these artifacts and damage include low-dose imaging techniques, which minimize electron fluence to below damage thresholds—typically 10-20 electrons per Ų—while employing computational averaging to recover signal from noisy data. Cryo-cooling preserves sample integrity by reducing molecular motion and damage rates by several-fold compared to room temperature, enabling imaging of hydrated, native-state structures with minimal alteration.

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