transmission electron microscopy transmission electron microscopy 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 sensor such as a scintillator attached to a charge-coupled device. a tem image of a cluster of poliovirus. the polio virus is 30 nm in diameter. 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. history initial development the duplicate an early tem on display at the deutsches museum in munich, germany a transmission electron microscope (1976). 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. 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. in 1858, plücker observed the deflection of "cathode rays" (electrons) by magnetic fields. this effect was used by ferdinand braun in 1897 to build simple cathode-ray oscilloscope (cro) measuring devices. in 1891, 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. 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. 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 publication of the de broglie hypothesis in 1927. 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 volt the wavelength is already as short as 1.23 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. 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 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, 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. 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. 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. 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, with albert crewe at the university of chicago developing the field emission gun 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. 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. d = λ 2 n sin ⁡ α ≈ λ 2 na \approx 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 (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) the wavelength is λ e = h 2 m 0 e ( 1 + e 2 m 0 c 2 ) =e\left(1+c^\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 layout of optical components in a basic tem hairpin style tungsten filament on an insulating base. single crystal lab6 filament 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. 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. 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