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 byImproving resolution
At the time, electrons were understood to be charged particles of matter; the wave nature of electrons was not fully realized until the publication of theFurther 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. 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. 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, withBackground
Electrons
Theoretically, the maximum resolution, ''d'', that one can obtain with a light microscope has been limited by the wavelength of the photons (λ) that are being used to probe the sample and the numerical aperture ''NA'' of the system. : 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). 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 (as theorized byElectron source
From the top down, the TEM consists of an emission source or cathode, which may be a tungsten filament or needle, or a lanthanum hexaboride ( LaB6) single crystal source. The gun is connected to a high voltage source (typically ~100–300 kV) and, given sufficient current, the gun will begin to emit electrons either by thermionic or field electron emission into the vacuum. In the case of a thermionic source, the electron source is typically 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 often collectively called the "electron gun". After it leaves the gun, the beam is typically accelerated by a series of electrostatic plates 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. 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 for electromagnets to manipulate the electron beam. The use of magnetic fields allows for the formation of a magnetic lens of variable focusing power, the lens shape originating due to the distribution of magnetic flux. Additionally,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 back up to get an image on a camera. 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 simply by adjusting the current passing through the coils. This provides a flexibility of operation that gets further multiplied when the lenses are assembled into stacks of independent lenses, each of which can focus, defocus, magnify, and/or collimate the beam coming from the previous lens. This allows a single lens system, between the source and the sample (the "condenser lens" system) to produce a parallel beam over 1 millimeter in diameter, a tightly focused beam smaller than an atom, or anything in between. An additional lens stack, the "intermediate/projector" lens system, is after the sample. It can be adjusted to produce a focused diffraction pattern or image of the sample with a magnification varying over a huge range. Many single microscopes can cover the magnification range from roughly 100X to more than 1,000,000X. Equally important to the lenses are the apertures. These are circular holes in thin strips of heavy metal, placed at well-chosen points in the column of lenses. Some are fixed in size and position and play important roles in limiting x-ray generation and improving the vacuum performance. They also prevent electrons from passing through the outermost parts of the magnetic lenses which, due to large lens aberrations, focus the electron beams extremely poorly. Others can be freely switched among several different sizes and have their positions adjusted. These "variable apertures" are used to determine the beam current reaching the sample and also to improve the ability to focus the beam. Variable apertures after the sample position further 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. Skillfully used, these apertures allow remarkably precise and detailed study of the defects in crystals. The electron-optical system also includes deflectors and stigmators, usually made of small electromagnets. Unlike the lenses, the magnetic fields produced by the deflectors are oriented primarily to deflect the beam and not to focus it. The deflectors allow the position and angle of the beam at the sample position to be independently controlled (as is essential for STEM) and also ensure that the beams remain near the low-aberration centers of every lens in the lens stacks. The stigmators provide an auxiliary fine focusing, compensating 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. AdditionalReciprocity
The optical reciprocity theorem, or principle of Helmholtz reciprocity, generally holds true for elastically scattered electrons in an absorbing medium, as is often the case under standard TEM operating conditions. 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. 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. In a TEM, electromagnetic lenses have been shown not to interfere noticeably with observations of reciprocity, provided that elastic scattering processes dominate in the sample and the sample is not strongly magnetic. Careful application of the reciprocity theorem in cases where it is valid give a TEM user considerable flexibility in taking and interpreting images and electron diffraction patterns. Reciprocity may also be 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. Imaging systems in a TEM consist of aComponents
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. 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 orSpecimen 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. 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 orElectron 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 theElectron 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, 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. 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.Apertures
Apertures are annular metallic plates, through which electrons that are further than a fixed distance from the optic axis may be excluded. These consist of a small metallic disc that is sufficiently thick to prevent electrons from passing through the disc, whilst permitting axial electrons. This permission of central electrons in a TEM causes two effects simultaneously: firstly, apertures decrease the beam intensity as electrons are filtered from the beam, which may be desired in the case of beam sensitive samples. Secondly, this filtering removes electrons that are scattered to high angles, which may be due to unwanted processes such as spherical or chromatic aberration, or due to diffraction from interaction within the sample. Apertures are either a fixed aperture within the column, such as at the condenser lens, or are a movable aperture, which can be inserted or withdrawn from the beam path, or moved in the plane perpendicular to the beam path. Aperture assemblies are mechanical devices which allow for the selection of different aperture sizes, which may be used by the operator to trade off intensity and the filtering effect of the aperture. Aperture assemblies are often equipped with micrometers to move the aperture, required during optical calibration.Imaging methods
Imaging methods in TEM use the information contained in the electron waves exiting from the sample to form an image. The projector lenses allow for the correct positioning of this electron wave distribution onto the viewing system. The observed intensity, ''I'', of the image, assuming sufficiently high quality of imaging device, can be approximated as proportional to the time-averaged squared absolute value of the amplitude of the electron wavefunctions, where the wave that forms the exit beam is denoted by Ψ. : Different imaging methods therefore attempt to modify the electron waves exiting the sample in a way that provides information about the sample, or the beam itself. From the previous equation, it can be deduced that the observed image depends not only on the amplitude of beam, but also on the phase of the electrons, although phase effects may often be ignored at lower magnifications. Higher resolution imaging requires thinner samples and higher energies of incident electrons, which means that the sample can no longer be considered to be absorbing electrons (i.e., via a Beer's law effect). Instead, the sample can be modeled as an object that does not change the amplitude of the incoming electron wave function, but instead modifies the phase of the incoming wave; in this model, the sample is known as a pure phase object. For sufficiently thin specimens, phase effects dominate the image, complicating analysis of the observed intensities. To improve the contrast in the image, the TEM may be operated at a slight defocus to enhance contrast, owing to convolution by theContrast formation
The contrast between two adjacent areas in a TEM image can be defined as the difference in the electron densities in image plane. Due to the scattering of the incident beam by the sample, the amplitude and phase of the electron wave change, which results in ''amplitude contrast'' and ''phase contrast'', correspondingly. Most images have both contrast components. Amplitude–contrast is obtained due to removal of some electrons before the image plane. During their interaction with the specimen some of electrons will be lost due to absorption, or due to scattering at very high angles beyond the physical limitation of microscope or are blocked by the objective aperture. While the first two losses are due to the specimen and microscope construction, the objective aperture can be used by operator to enhance the contrast. Figure on the right shows a TEM image (a) and the corresponding diffraction pattern (b) of Pt polycrystalline film taken without an objective aperture. In order to enhance the contrast in the TEM image the number of scattered beams as visible in the diffraction pattern should be reduced. This can be done by selecting a certain area in the back focal plane such as only the central beam or a specific diffracted beam (angle), or combinations of such beams. By intentionally selecting an objective aperture which only permits the non-diffracted beam to pass beyond the back focal plane (and onto the image plane): one creates a Bright-Field (BF) image (c), whereas if the central, non-diffracted beam is blocked: one may obtain Dark-Field (DF) images such as those shown in (d-e). The DF images (d-e) were obtained by selecting the diffracted beams indicated in diffraction pattern with circles (b) using an aperture at the back focal plane. Grains from which electrons are scattered into these diffraction spots appear brighter. More details about diffraction contrast formation are given further. There are two types of amplitude contrast – mass–thickness and diffraction contrast. First, let's consider ''mass–thickness contrast''. When the beam illuminates two neighbouring areas with low mass (or thickness) and high mass (or thickness), the heavier region scatters electrons at bigger angles. These strongly scattered electrons are blocked in BF TEM mode by objective aperture. As a result, heavier regions appear darker in BF images (have low intensity). Mass–thickness contrast is most important for non–crystalline, amorphous materials. ''Diffraction contrast'' occurs due to a specific crystallographic orientation of a grain. In such a case the crystal is in a so-called Bragg condition, whereby atomic planes are oriented in a way that there is a high probability of scattering. Thus diffraction contrast provides information on the orientation of the crystals in a polycrystalline sample. Note that in case diffraction contrast exists, the contrast cannot be interpreted as due to mass or thickness variations.Diffraction contrast
Samples can exhibit diffraction contrast, whereby the electron beam undergoes Bragg scattering, which in the case of a crystalline sample, disperses electrons into discrete locations in the back focal plane. By the placement of apertures in the back focal plane, i.e. the objective aperture, the desired Bragg reflections can be selected (or excluded), thus only parts of the sample that are causing the electrons to scatter to the selected reflections will end up projected onto the imaging apparatus. If the reflections that are selected do not include the unscattered beam (which will appear up at the focal point of the lens), then the image will appear dark wherever no sample scattering to the selected peak is present, as such a region without a specimen will appear dark. This is known as a dark-field image. Modern TEMs are often equipped with specimen holders that allow the user to tilt the specimen to a range of angles in order to obtain specific diffraction conditions, and apertures placed above the specimen allow the user to select electrons that would otherwise be diffracted in a particular direction from entering the specimen. Applications for this method include the identification ofPhase contrast
Crystal structure can also be investigated by high-resolution transmission electron microscopy (HRTEM), also known as phase contrast. When using a field emission source and a specimen of uniform thickness, the images are formed due to differences in phase of electron waves, which is caused by specimen interaction. Image formation is given by the complex modulus of the incoming electron beams. As such, the image is not only dependent on the number of electrons hitting the screen, making direct interpretation of phase contrast images more complex. However this effect can be used to an advantage, as it can be manipulated to provide more information about the sample, such as in complex phase retrieval techniques.Diffraction
As previously stated, by adjusting the magnetic lenses such that the back focal plane of the lens rather than the imaging plane is placed on the imaging apparatus a diffraction pattern can be generated. For thin crystalline samples, this produces an image that consists of a pattern of dots in the case of a single crystal, or a series of rings in the case of a polycrystalline or amorphous solid material. For the single crystal case the diffraction pattern is dependent upon the orientation of the specimen and the structure of the sample illuminated by the electron beam. This image provides the investigator with information about the space group symmetries in the crystal and the crystal's orientation to the beam path. This is typically done without using any information but the position at which the diffraction spots appear and the observed image symmetries. Diffraction patterns can have a large dynamic range, and for crystalline samples, may have intensities greater than those recordable by CCD. As such, TEMs may still be equipped with film cartridges for the purpose of obtaining these images, as the film is a single use detector. Analysis of diffraction patterns beyond point-position can be complex, as the image is sensitive to a number of factors such as specimen thickness and orientation, objective lens defocus, and spherical and chromatic aberration. Although quantitative interpretation of the contrast shown in lattice images is possible, it is inherently complicated and can require extensive computer simulation and analysis, such as electron multislice analysis. More complex behaviour in the diffraction plane is also possible, with phenomena such as Kikuchi lines arising from multiple diffraction within the crystalline lattice. InElectron energy loss spectroscopy (EELS)
Using the advanced technique of electron energy loss spectroscopy (EELS), for TEMs appropriately equipped, electrons can be separated into a spectrum based upon their velocity (which is closely related to their kinetic energy, and thus energy loss from the beam energy), usingThree-dimensional imaging
As TEM specimen holders typically allow for the rotation of a sample by a desired angle, multiple views of the same specimen can be obtained by rotating the angle of the sample along an axis perpendicular to the beam. By taking multiple images of a single TEM sample at differing angles, typically in 1° increments, a set of images known as a "tilt series" can be collected. This methodology was proposed in the 1970s bySample preparation
Sample preparation in TEM can be a complex procedure. TEM specimens should be less than 100 nanometers thick for a conventional TEM. UnlikeTissue sectioning
Biological tissue is often embedded in a resin block then thinned to less than 100 nm on anSample staining
TEM samples of biological tissues need high atomic number stains to enhance contrast. The stain absorbs the beam electrons or scatters part of the electron beam which otherwise is projected onto the imaging system. Compounds of heavy metals such as osmium, lead, uranium orMechanical milling
Mechanical polishing is also used to prepare samples for imaging on the TEM. Polishing needs to be done to a high quality, to ensure constant sample thickness across the region of interest. A diamond, or cubic boron nitride polishing compound may be used in the final stages of polishing to remove any scratches that may cause contrast fluctuations due to varying sample thickness. Even after careful mechanical milling, additional fine methods such as ion etching may be required to perform final stage thinning.Chemical etching
Certain samples may be prepared by chemical etching, particularly metallic specimens. These samples are thinned using a chemical etchant, such as an acid, to prepare the sample for TEM observation. Devices to control the thinning process may allow the operator to control either the voltage or current passing through the specimen, and may include systems to detect when the sample has been thinned to a sufficient level of optical transparency.Ion etching
Ion etching is a sputtering process that can remove very fine quantities of material. This is used to perform a finishing polish of specimens polished by other means. Ion etching uses an inert gas passed through an electric field to generate a plasma stream that is directed to the sample surface. Acceleration energies for gases such as argon are typically a few kilovolts. The sample may be rotated to promote even polishing of the sample surface. The sputtering rate of such methods is on the order of tens of micrometers per hour, limiting the method to only extremely fine polishing. Ion etching by argon gas has been recently shown to be able to file down MTJ stack structures to a specific layer which has then been atomically resolved. The TEM images taken in plan view rather than cross-section reveal that the MgO layer within MTJs contains a large number of grain boundaries that may be diminishing the properties of devices.Ion milling ( FIB)
More recently focused ion beam methods have been used to prepare samples. FIB is a relatively new technique to prepare thin samples for TEM examination from larger specimens. Because FIB can be used to micro-machine samples very precisely, it is possible to mill very thin membranes from a specific area of interest in a sample, such as a semiconductor or metal. Unlike inert gas ion sputtering, FIB makes use of significantly more energetic gallium ions and may alter the composition or structure of the material through gallium implantation.Nanowire assisted transfer
For a minimal introduction of stress and bending to transmission electron microscopy (TEM) samples ( lamellae, thin films, and other mechanically and beam sensitive samples), when transferring inside a focused ion beam (FIB), flexible metallic nanowires can be attached to a typically rigid micromanipulator. The main advantages of this method include a significant reduction of sample preparation time (quick welding and cutting of nanowire at low beam current), and minimization of stress-induced bending, Pt contamination, and ion beam damage. This technique is particularly suitable for in situ electron microscopy sample preparation.Replication
Samples may also be replicated using cellulose acetate film, the film subsequently coated with a heavy metal such as platinum, the original film dissolved away, and the replica imaged on the TEM. Variations of the replica technique are used for both materials and biological samples. In materials science a common use is for examining the fresh fracture surface of metal alloys.Modifications
The capabilities of the TEM can be further extended by additional stages and detectors, sometimes incorporated on the same microscope.Scanning TEM
A TEM can be modified into a scanning transmission electron microscope (STEM) by the addition of a system that rasters a convergent beam across the sample to form the image, when combined with suitable detectors. Scanning coils are used to deflect the beam, such as by an electrostatic shift of the beam, where the beam is then collected using a current detector such as a Faraday cup, which acts as a direct electron counter. By correlating the electron count to the position of the scanning beam (known as the "probe"), the transmitted component of the beam may be measured. The non-transmitted components may be obtained either by beam tilting or by the use of annular dark field detectors. Fundamentally, TEM and STEM are linked via Helmholtz reciprocity. A STEM is a TEM in which the electron source and observation point have been switched relative to the direction of travel of the electron beam. See the ray diagrams in the figure on the right. The STEM instrument effectively relies on the same optical set-up as a TEM, but operates by flipping the direction of travel of the electrons (or reversing time) during operation of a TEM. Rather than using an aperture to control detected electrons, as in TEM, a STEM uses various detectors with collection angles that may be adjusted depending on which electrons the user wants to capture.Low-voltage electron microscope
A low-voltage electron microscope (LVEM) is operated at relatively low electron accelerating voltage between 5–25 kV. Some of these can be a combination of SEM, TEM and STEM in a single compact instrument. Low voltage increases image contrast which is especially important for biological specimens. This increase in contrast significantly reduces, or even eliminates the need to stain. Resolutions of a few nm are possible in TEM, SEM and STEM modes. The low energy of the electron beam means that permanent magnets can be used as lenses and thus a miniature column that does not require cooling can be used.Cryo-TEM
Cryogenic transmission electron microscopy (Cryo-TEM) uses a TEM with a specimen holder capable of maintaining the specimen at liquid nitrogen or liquid helium temperatures. This allows imaging specimens prepared in vitreous ice, the preferred preparation technique for imaging individual molecules or macromolecular assemblies, imaging of vitrified solid-electrolye interfaces, and imaging of materials that are volatile in high vacuum at room temperature, such as sulfur.Environmental/In-situ TEM
In-situ experiments may also be conducted in TEM using differentially pumped sample chambers, or specialized holders. Types of in-situ experiments include studying nanomaterials, biological specimens, chemical reactions of molecules,High Temperature In-Situ TEM
Many phase transformations occur during heating. Additionally, coarsening and grain growth, along with other diffusion-related processes occur more rapidly at elevated temperatures, where kinetics are improved, allowing for the observation of related phenomena under transmission electron microscopy within reasonable time scales. This also allows for the observation of phenomena that occur at elevated temperatures and disappear or are not uniformly preserved in ex-situ samples. High temperature TEM introduces various additional challenges which must be addressed in the mechanics of high temperature holders, including but not limited to drift-correction, temperature measurement, and decreased spatial resolution at the expense of more complex holders. Sample drift in the TEM is linearly proportional to the temperature differential between the room and holder. With temperatures as high as 1500C in modern holders, samples may experience significant drift and vertical displacement (bulging), requiring continuous focus or stage adjustments, inducing resolution loss and mechanical drift. Individual labs and manufacturers have developed software coupled with advanced cooling systems to correct for thermal drift based on the predicted temperature in the sample chamber These systems often take 30 min-many hours for sample shifts to stabilize. While significant progress has been made, no universal TEM attachment has been made to account for drift at elevated temperatures. An additional challenge of many of these specialized holders is knowing the local sample temperature. Many high temperature holders utilize a tungsten filament to locally heat the sample. Ambiguity in temperature in furnace heaters (W wire) with thermocouples arises from the thermal contact between the furnace and the TEM grid; complicated by temperature gradients along the sample caused by varying thermal conductivity with different samples and grid materials. With different holders both commercial and lab made, different methods for creating temperature calibration are available. Manufacturers like Gatan use IR pyrometry to measure temperature gradients over their entire sample. An even better method to calibrate is Raman spectroscopy which measures the local temperature of Si powder on electron transparent windows and quantitatively calibrates the IR pyrometry. These measurements have guaranteed accuracy within 5%. Research laboratories have also performed their own calibrations on commercial holders. Researchers at NIST utilized Raman spectroscopy to map the temperature profile of a sample on a TEM grid and achieve very precise measurements to enhance their research. Similarly, a research group in Germany utilized X-ray diffraction to measure slight shifts in lattice spacing caused by changes in temperature to back calculate the exact temperature in the holder. This process required careful calibration and exact TEM optics. Other examples include the use of EELS to measure local temperature using change of gas density, and resistivity changes. Optimal resolution in a TEM is achieved when spherical aberrations are corrected with objective lens. However, due to the geometry of most TEMs, inserting large in-situ holders requires the user to compromise the objective lens and endure spherical aberrations. Therefore, there is a compromise between the width of the pole-piece gap and spatial resolution below 0.1 nm. Research groups at various institutions have tried to overcome spherical aberrations through use of monochromators to achieve 0.05 nm resolution with a 5 mm pole piece gap.In-Situ Mechanical TEM
Mechanical testing inside TEM columns might help understand phenomena that happen while straining samples, such as necking. Multiple methods of mechanical testing can be conducted inside a TEM. Current instruments have the ability to place samples under tension, compression, shearing and bending. One of the pioneers of this technique was Dr. Heinz G.F. Wilsdorf, who conducted a tension test inside a TEM in 1958. They used a screw to actuate the sample and place it under stress, however these first tests were not yet able to compress or bend the material. Modern TEM sample holders have been developed to conduct multiple mechanical tests beyond just tension for in situ studies. Microelectromechanical systems (MEMs) have further enhanced the sample holder's ability to conduct mechanical experiments. They provide higher resolution for handling TEM samples, leading to better results. More than just qualitative understanding of the sample, in 2017, Dr. Han developed a method of using MEMs holders to do qualitative investigation under mechanical stresses. In Situ TEM can also be coupled with other standard TEM practices such as EELS and XEDS to acquire more information on the sample structures.Aberration corrected TEM
Modern research TEMs may include aberration correctors, to reduce the amount of distortion in the image. Incident beam monochromators may also be used which reduce the energy spread of the incident electron beam to less than 0.15 eV. Major aberration corrected TEM manufacturers include JEOL, Hitachi High-technologies, FEI Company, and NION.Ultrafast and dynamic TEM
It is possible to reach temporal resolution far beyond that of the readout rate of electron detectors with the use of pulsed electrons. Pulses can be produced by either modifying the electron source to enable laser-triggered photoemission or by installation of an ultrafast beam blanker. This approach is termed ultrafast transmission electron microscopy when stroboscopic pump-probe illumination is used: an image is formed by the accumulation of many ultrashort electron pulses (typically of hundreds of femtoseconds) with a fixed time delay between the arrival of the electron pulse and the sample excitation. On the other hand, the use of single or a short sequence of electron pulses with a sufficient number of electrons to form an image from each pulse is called dynamic transmission electron microscopy. Temporal resolution down to hundreds of femtoseconds and spatial resolution comparable to that available with a Schottky field emission source is possible in ultrafast TEM, but the technique can only image reversible processes that can be reproducibly triggered millions of times. Dynamic TEM can resolve irreversible processes down to tens of nanoseconds and tens of nanometers. The technique has been pioneered at the early 2000s in laboratories in Germany (Limitations
There are a number of drawbacks to the TEM technique. Many materials require extensive sample preparation to produce a sample thin enough to be electron transparent, which makes TEM analysis a relatively time-consuming process with a low throughput of samples. The structure of the sample may also be changed during the preparation process. Also the field of view is relatively small, raising the possibility that the region analyzed may not be characteristic of the whole sample. There is potential that the sample may be damaged by the electron beam, particularly in the case of biological materials.Resolution limits
The limit of resolution obtainable in a TEM may be described in several ways, and is typically referred to as the information limit of the microscope. One commonly used value is a cut-off value of theSee also
* Electron microscope *References
External links