Transmission electron microscopy (TEM) is a microscopy technique in which a beam of electrons is transmitted through a specimen to form an image. The specimen is most often an ultrathin section less than 100 nm thick or a suspension on a grid. An image is formed from the interaction of the electrons with the sample as the beam is transmitted through the specimen. The image is then magnified and focused onto an imaging device, such as a fluorescent screen, a layer of photographic film, or a detector such as a scintillator attached to a charge-coupled device or a direct electron detector.

Transmission electron microscopes are capable of imaging at a significantly higher resolution than light microscopes, owing to the smaller de Broglie wavelength of electrons. This enables the instrument to capture fine detail—even as small as a single column of atoms, which is thousands of times smaller than a resolvable object seen in a light microscope. Transmission electron microscopy is a major analytical method in the physical, chemical and biological sciences. TEMs find application in cancer research, virology, and materials science as well as pollution, nanotechnology and semiconductor research, but also in other fields such as paleontology and palynology.

TEM instruments have multiple operating modes including conventional imaging, scanning TEM imaging (STEM), diffraction, spectroscopy, and combinations of these. Even within conventional imaging, there are many fundamentally different ways that contrast is produced, called "image contrast mechanisms". Contrast can arise from position-to-position differences in the thickness or density ("mass-thickness contrast"), atomic number ("Z contrast", referring to the common abbreviation Z for atomic number), crystal structure or orientation ("crystallographic contrast" or "diffraction contrast"), the slight quantum-mechanical phase shifts that individual atoms produce in electrons that pass through them ("phase contrast"), the energy lost by electrons on passing through the sample ("spectrum imaging") and more. Each mechanism tells the user a different kind of information, depending not only on the contrast mechanism but on how the microscope is used—the settings of lenses, apertures, and detectors. What this means is that a TEM is capable of returning an extraordinary variety of nanometer- and atomic-resolution information, in ideal cases revealing not only where all the atoms are but what kinds of atoms they are and how they are bonded to each other. For this reason TEM is regarded as an essential tool for nanoscience in both biological and materials fields.

The first TEM was demonstrated by Max Knoll and Ernst Ruska in 1931, with this group developing the first TEM with resolution greater than that of light in 1933 and the first commercial TEM in 1939. In 1986, Ruska was awarded the Nobel Prize in physics for the development of transmission electron microscopy.^[2]

History

Initial development

In 1873, Ernst Abbe proposed that the ability to resolve detail in an object was limited approximately by the wavelength of the light used in imaging or a few hundred nanometers for visible light microscopes. Developments in ultraviolet (UV) microscopes, led by Köhler and Rohr, increased resolving power by a factor of two.^[3] However this required expensive quartz optics, due to the absorption of UV by glass. It was believed that obtaining an image with sub-micrometer information was not possible due to this wavelength constraint.^[4]

In 1858, Plücker observed the deflection of "cathode rays" (electrons) by magnetic fields.^[5] This effect was used by Ferdinand Braun in 1897 to build simple cathode-ray oscilloscope (CRO) measuring devices.^[6] In 1891, Eduard Riecke noticed that the cathode rays could be focused by magnetic fields, allowing for simple electromagnetic lens designs. In 1926, Hans Busch published work extending this theory and showed that the lens maker's equation could, with appropriate assumptions, be applied to electrons.^[2]

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 the CRO design. The team consisted of several PhD students including Ernst Ruska and Bodo von Borries. The research team worked on lens design and CRO column placement, to optimize parameters to construct better CROs, and make electron optical components to generate low magnification (nearly 1:1) images. In 1931, the group successfully generated magnified images of mesh grids placed over the anode aperture. The device used two magnetic lenses to achieve higher magnifications, arguably creating the first electron microscope. In that same year, Reinhold Rudenberg, the scientific director of the Siemens company, patented an electrostatic lens electron microscope.^[4][7]

Improving resolution

At the time, electrons were understood to be charged particles of matter; the wave nature of electrons was not fully realized until the PhD thesis of Louis de Broglie in 1924.^[8] Knoll's research group was unaware of this publication until 1932, when they realized that the de Broglie wavelength of electrons was many orders of magnitude smaller than that for light, theoretically allowing for imaging at atomic scales. (Even for electrons with a kinetic energy of just 1 electronvolt the wavelength is already as short as 1.18 nm.) In April 1932, Ruska suggested the construction of a new electron microscope for direct imaging of specimens inserted into the microscope, rather than simple mesh grids or images of apertures. With this device successful diffraction and normal imaging of an aluminium sheet was achieved. However the magnification achievable was lower than with light microscopy. Magnifications higher than those available with a light microscope were achieved in September 1933 with images of cotton fibers quickly acquired before being damaged by the electron beam.^[4]

At this time, interest in the electron microscope had increased, with other groups, such as that of Paul Anderson and Kenneth Fitzsimmons of Washington State University^[9] and that of Albert Prebus and James Hillier at the University of Toronto, who constructed the first TEMs in North America in 1935 and 1938, respectively,^[10] continually advancing TEM design.

Research continued on the electron microscope at Siemens in 1936, where the aim of the research was the development and improvement of TEM imaging properties, particularly with regard to biological specimens. At this time electron microscopes were being fabricated for specific groups, such as the "EM1" device used at the UK National Physical Laboratory.^[11] In 1939, the first commercial electron microscope, pictured, was installed in the Physics department of IG Farben-Werke. Further work on the electron microscope was hampered by the destruction of a new laboratory constructed at Siemens by an air raid, as well as the death of two of the researchers, Heinz Müller and Friedrick Krause during World War II.^[12]

Further research

After World War II, Ruska resumed work at Siemens, where he continued to develop the electron microscope, producing the first microscope with 100k magnification.^[12] The fundamental structure of this microscope design, with multi-stage beam preparation optics, is still used in modern microscopes. The worldwide electron microscopy community advanced with electron microscopes being manufactured in Manchester UK, the USA (RCA), Germany (Siemens) and Japan (JEOL). The first international conference in electron microscopy was in Delft in 1949, with more than one hundred attendees.^[11] Later conferences included the "First" international conference in Paris, 1950 and then in London in 1954.

With the development of TEM, the associated technique of scanning transmission electron microscopy (STEM) was re-investigated and remained undeveloped until the 1970s, with Albert Crewe at the University of Chicago developing the field emission gun^[13] and adding a high quality objective lens to create the modern STEM. Using this design, Crewe demonstrated the ability to image atoms using annular dark-field imaging. Crewe and coworkers at the University of Chicago developed the cold field electron emission source and built a STEM able to visualize single heavy atoms on thin carbon substrates.^[14]

Background

Electrons

Theoretically, the maximum resolution, d, that one can obtain with a light microscope is limited by the wavelength of the photons (λ) and the numerical aperture NA of the system.^[15]

${\displaystyle d={\frac {\lambda }{2n\sin \alpha }}\approx {\frac {\lambda }{2\,{\textrm {NA}}}}}$

where n is the index of refraction of the medium in which the lens is working and α is the maximum half-angle of the cone of light that can enter the lens (see numerical aperture).^[16] Early twentieth century scientists theorized ways of getting around the limitations of the relatively large wavelength of visible light (wavelengths of 400–700 nanometers) by using electrons. Like all matter, electrons have both wave and particle properties (matter wave), and their wave-like properties mean that a beam of electrons can be focused and diffracted much like light can. The wavelength of electrons is related to their kinetic energy via the de Broglie equation, which says that the wavelength is inversely proportional to the momentum. Taking into account relativistic effects (as in a TEM an electron's velocity is a substantial fraction of the speed of light, c^[17]) the wavelength is

${\displaystyle \lambda _{e}={\frac {h}{\sqrt {2m_{0}E\left(1+{\frac {E}{2m_{0}c^{2}}}\right)}}}}$

where, h is Planck's constant, m₀ is the rest mass of an electron and E is the kinetic energy of the accelerated electron.

Electron source

From the top down, the TEM consists of an emission source or cathode, which may be a tungsten filament, a lanthanum hexaboride (LaB₆) single crystal or a field emission gun.^[18] The gun is connected to a high voltage source (typically ~100–300 kV) and emits electrons either by thermionic or field electron emission into the vacuum. In the case of a thermionic source, the electron source is mounted in a Wehnelt cylinder to provide preliminary focus of the emitted electrons into a beam while also stabilizing the current using a passive feedback circuit. A field emission source uses instead electrostatic electrodes called an extractor, a suppressor, and a gun lens, with different voltages on each, to control the electric field shape and intensity near the sharp tip. The combination of the cathode and these first electrostatic lens elements is collectively called the "electron gun". After it leaves the gun, the beam is typically accelerated until it reaches its final voltage and enters the next part of the microscope: the condenser lens system. These upper lenses of the TEM then further focus the electron beam to the desired size and location on the sample.^[19]

Manipulation of the electron beam is performed using two physical effects. The interaction of electrons with a magnetic field will cause electrons to move according to the left hand rule, thus allowing electromagnets to manipulate the electron beam. Additionally, electrostatic fields can cause the electrons to be deflected through a constant angle. Coupling of two deflections in opposing directions with a small intermediate gap allows for the formation of a shift in the beam path, allowing for beam shifting.

Optics

The lenses of a TEM are what gives it its flexibility of operating modes and ability to focus beams down to the atomic scale and magnify them to get an image. A lens is usually made of a solenoid coil nearly surrounded by ferromagnetic materials designed to concentrate the coil's magnetic field into a precise, confined shape. When an electron enters and leaves this magnetic field, it spirals around the curved magnetic field lines in a way that acts very much as an ordinary glass lens does for light—it is a converging lens. But, unlike a glass lens, a magnetic lens can very easily change its focusing power by adjusting the current passing through the coils.

Equally important to the lenses are the apertures. These are circular holes in thin strips of heavy metal. Some are fixed in size and position and play important roles in limiting x-ray generation and improving the vacuum performance. Others can be freely switched among several different sizes and have their positions adjusted. Variable apertures after the sample allow the user to select the range of spatial positions or electron scattering angles to be used in the formation of an image or a diffraction pattern.

The electron-optical system also includes deflectors and stigmators, usually made of small electromagnets. The deflectors allow the position and angle of the beam at the sample position to be independently controlled and also ensure that the beams remain near the low-aberration centers of every lens in the lens stacks. The stigmators compensate for slight imperfections and aberrations that cause astigmatism—a lens having a different focal strength in different directions.

Typically a TEM consists of three stages of lensing. The stages are the condenser lenses, the objective lenses, and the projector lenses. The condenser lenses are responsible for primary beam formation, while the objective lenses focus the beam that comes through the sample itself (in STEM scanning mode, there are also objective lenses above the sample to make the incident electron beam convergent). The projector lenses are used to expand the beam onto the phosphor screen or other imaging device, such as film. The magnification of the TEM is due to the ratio of the distances between the specimen and the objective lens' image plane.^[20] TEM optical configurations differ significantly with implementation, with manufacturers using custom lens configurations, such as in spherical aberration corrected instruments,^[19] or TEMs using energy filtering to correct electron chromatic aberration.

Reciprocity

The optical reciprocity theorem, or principle of Helmholtz reciprocity, generally holds true for elastically scattered electrons, as is often the case under standard TEM operating conditions.^[21][22] The theorem states that the wave amplitude at some point B as a result of electron point source A would be the same as the amplitude at A due to an equivalent point source placed at B.^[22] Simply stated, the wave function for electrons focused through any series of optical components that includes only scalar (i.e. not magnetic) fields will be exactly equivalent if the electron source and observation point are reversed. R

Reciprocity is used to understand scanning transmission electron microscopy (STEM) in the familiar context of TEM, and to obtain and interpret images using STEM.

Display and detectors

The key factors when considering electron detection include detective quantum efficiency (DQE), point spread function (PSF), modulation transfer function (MTF), pixel size and array size, noise, data readout speed, and radiation hardness.^[23]

Imaging systems in a TEM consist of a phosphor screen, which may be made of fine (10–100 μm) particulate zinc sulfide, for direct observation by the operator, and an image recording system such as photographic film,^[24] doped YAG screen coupled CCDs,^[25] or other digital detector.^[23] Typically these devices can be removed or inserted into the beam path as required. (Photograph film is no longer used.) The first report of using a Charge-Coupled Device (CCD) detector for TEM was in 1982,^[26] but the technology didn't find widespread use until the late 1990s/early 2000s.^[27] Monolithic active-pixel sensors (MAPSs) were also used in TEM.^[28] CMOS detectors, which are faster and more resistant to radiation damage than CCDs, have been used for TEM since 2005.^[29][30] In the early 2010s, further development of CMOS technology allowed for the detection of single electron counts ("counting mode").^[31][32] These Direct Electron Detectors are available from Gatan, FEI, Quantum Detectors and Direct Electron.^[28]

Components

A TEM is composed of several components, which include a vacuum system in which the electrons travel, an electron emission source for generation of the electron stream, a series of electromagnetic lenses, as well as electrostatic plates. The latter two allow the operator to guide and manipulate the beam as required. Also required is a device to allow the insertion into, motion within, and removal of specimens from the beam path. Imaging devices are subsequently used to create an image from the electrons that exit the system.

Vacuum system

To increase the mean free path of the electron gas interaction, a standard TEM is evacuated to low pressures, typically on the order of 10⁻⁴ Pa.^[33] The need for this is twofold: first the allowance for the voltage difference between the cathode and the ground without generating an arc, and secondly to reduce the collision frequency of electrons with gas atoms to negligible levels—this effect is characterized by the mean free path. TEM components such as specimen holders and film cartridges must be routinely inserted or replaced requiring a system with the ability to re-evacuate on a regular basis. As such, TEMs are equipped with multiple pumping systems and airlocks and are not permanently vacuum sealed.

The vacuum system for evacuating a TEM to an operating pressure level consists of several stages. Initially, a low or roughing vacuum is achieved with either a rotary vane pump or diaphragm pumps setting a sufficiently low pressure to allow the operation of a turbo-molecular or diffusion pump establishing high vacuum level necessary for operations. To allow for the low vacuum pump to not require continuous operation, while continually operating the turbo-molecular pumps, the vacuum side of a low-pressure pump may be connected to chambers which accommodate the exhaust gases from the turbo-molecular pump.^[34] Sections of the TEM may be isolated by the use of pressure-limiting apertures to allow for different vacuum levels in specific areas such as a higher vacuum of 10⁻⁴ to 10⁻⁷ Pa or higher in the electron gun in high-resolution or field-emission TEMs.

High-voltage TEMs require ultra-high vacuums on the range of 10⁻⁷ to 10⁻⁹ Pa to prevent the generation of an electrical arc, particularly at the TEM cathode.^[35] As such for higher voltage TEMs a third vacuum system may operate, with the gun isolated from the main chamber either by gate valves or a differential pumping aperture – a small hole that prevents the diffusion of gas molecules into the higher vacuum gun area faster than they can be pumped out. For these very low pressures, either an ion pump or a getter material is used.

Poor vacuum in a TEM can cause several problems ranging from the deposition of gas inside the TEM onto the specimen while viewed in a process known as electron beam induced deposition to more severe cathode damages caused by electrical discharge.^[35] The use of a cold trap to adsorb sublimated gases in the vicinity of the specimen largely eliminates vacuum problems that are caused by specimen sublimation.^[34]

Specimen stage

TEM specimen stage designs include airlocks to allow for insertion of the specimen holder into the vacuum with minimal loss of vacuum in other areas of the microscope. The specimen holders hold a standard size of sample grid or self-supporting specimen. Standard TEM grid sizes are 3.05 mm diameter, with a thickness and mesh size ranging from a few to 100 μm. The sample is placed onto the meshed area having a diameter of approximately 2.5 mm. Usual grid materials are copper, molybdenum, gold or platinum. This grid is placed into the sample holder, which is paired with the specimen stage. A wide variety of designs of stages and holders exist, depending upon the type of experiment being performed. In addition to 3.05 mm grids, 2.3 mm grids are sometimes, if rarely, used. These grids were particularly used in the mineral sciences where a large degree of tilt can be required and where specimen material may be extremely rare. Electron transparent specimens have a thickness usually less than 100 nm, but this value depends on the accelerating voltage.

Once inserted into a TEM, the sample has to be manipulated to locate the region of interest to the beam, such as in single grain diffraction, in a specific orientation. To accommodate this, the TEM stage allows movement of the sample in the XY plane, Z height adjustment, and commonly a single tilt direction parallel to the axis of side entry holders. Sample rotation may be available on specialized diffraction holders and stages. Some modern TEMs provide the ability for two orthogonal tilt angles of movement with specialized holder designs called double-tilt sample holders. Some stage designs, such as top-entry or vertical insertion stages once common for high resolution TEM studies, may simply only have X-Y translation available. The design criteria of TEM stages are complex, owing to the simultaneous requirements of mechanical and electron-optical constraints and specialized models are available for different methods.

A TEM stage is required to have the ability to hold a specimen and be manipulated to bring the region of interest into the path of the electron beam. As the TEM can operate over a wide range of magnifications, the stage must simultaneously be highly resistant to mechanical drift, with drift requirements as low as a few nm/minute while being able to move several μm/minute, with repositioning accuracy on the order of nanometers.^[36] Earlier designs of TEM accomplished this with a complex set of mechanical downgearing devices, allowing the operator to finely control the motion of the stage by several rotating rods. Modern devices may use electrical stage designs, using screw gearing in concert with stepper motors, providing the operator with a computer-based stage input, such as a joystick or trackball.

Two main designs for stages in a TEM exist, the side-entry and top entry version.^[25] Each design must accommodate the matching holder to allow for specimen insertion without either damaging delicate TEM optics or allowing gas into TEM systems under vacuum.

The most common is the side entry holder, where the specimen is placed near the tip of a long metal (brass or stainless steel) rod, with the specimen placed flat in a small bore. Along the rod are several polymer vacuum rings to allow for the formation of a vacuum seal of sufficient quality, when inserted into the stage. The stage is thus designed to accommodate the rod, placing the sample either in between or near the objective lens, dependent upon the objective design. When inserted into the stage, the side entry holder has its tip contained within the TEM vacuum, and the base is presented to atmosphere, the airlock formed by the vacuum rings.

Insertion procedures for side-entry TEM holders typically involve the rotation of the sample to trigger micro switches that initiate evacuation of the airlock before the sample is inserted into the TEM column.

The second design is the top-entry holder consists of a cartridge that is several cm long with a bore drilled down the cartridge axis. The specimen is loaded into the bore, possibly using a small screw ring to hold the sample in place. This cartridge is inserted into an airlock with the bore perpendicular to the TEM optic axis. When sealed, the airlock is manipulated to push the cartridge such that the cartridge falls into place, where the bore hole becomes aligned with the beam axis, such that the beam travels down the cartridge bore and into the specimen. Such designs are typically unable to be tilted without blocking the beam path or interfering with the objective lens.^[25]

Electron gun

The electron gun is formed from several components: the filament, a biasing circuit, a Wehnelt cap, and an extraction anode. By connecting the filament to the negative component power supply, electrons can be "pumped" from the electron gun to the anode plate and the TEM column, thus completing the circuit. The gun is designed to create a beam of electrons exiting from the assembly at some given angle, known as the gun divergence semi-angle, α. By constructing the Wehnelt cylinder such that it has a higher negative charge than the filament itself, electrons that exit the filament in a diverging manner are, under proper operation, forced into a converging pattern the minimum size of which is the gun crossover diameter.

The thermionic emission current density, J, can be related to the work function of the emitting material via Richardson's law

${\displaystyle J=AT^{2}\exp \left({\frac {-\Phi }{kT}}\right),}$

where A is the Richardson's constant, Φ is the work function and T is the temperature of the material.^[25]

This equation shows that in order to achieve sufficient current density it is necessary to heat the emitter, taking care not to cause damage by application of excessive heat. For this reason materials with either a high melting point, such as tungsten, or those with a low work function (LaB₆) are required for the gun filament.^[37] Furthermore, both lanthanum hexaboride and tungsten thermionic sources must be heated in order to achieve thermionic emission, this can be achieved by the use of a small resistive strip. To prevent thermal shock, there is often a delay enforced in the application of current to the tip, to prevent thermal gradients from damaging the filament, the delay is usually a few seconds for LaB₆, and significantly lower for tungsten^{[citation needed]}.

Electron lens

Electron lenses are designed to act in a manner emulating that of an optical lens, by focusing parallel electrons at some constant focal distance. Electron lenses may operate electrostatically or magnetically. The majority of electron lenses for TEM use electromagnetic coils to generate a convex lens. The field produced for the lens must be radially symmetrical, as deviation from the radial symmetry of the magnetic lens causes aberrations such as astigmatism, and worsens spherical and chromatic aberration. Electron lenses are manufactured from iron, iron-cobalt or nickel cobalt alloys,^[38] such as permalloy. These are selected for their magnetic properties, such as magnetic saturation, hysteresis and permeability.

The components include the yoke, the magnetic coil, the poles, the polepiece, and the external control circuitry. The pole piece must be manufactured in a very symmetrical manner, as this provides the boundary conditions for the magnetic field that forms the lens. Imperfections in the manufacture of the pole piece can induce severe distortions in the magnetic field symmetry, which induce distortions that will ultimately limit the lenses' ability to reproduce the object plane. The exact dimensions of the gap, pole piece internal diameter and taper, as well as the overall design of the lens is often performed by finite element analysis of the magnetic field, whilst considering the thermal and electrical constraints of the design.^[38]

The coils which produce the magnetic field are located within the lens yoke. The coils can contain a variable current, but typically use high voltages, and therefore require significant insulation in order to prevent short-circuiting the lens components. Thermal distributors are placed to ensure the extraction of the heat generated by the energy lost to resistance of the coil windings. The windings may be water-cooled, using a chilled water supply in order to facilitate the removal of the high thermal duty. Zdroj:https://en.wikipedia.org?pojem=Transmission_electron_microscopy
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