Transmission electron microscopy

Overview
Transmission electron microscopy (TEM) is an imaging technique whereby a beam of electrons is transmitted through a specimen, then an image is formed, magnified and directed to appear either on a fluorescent screen or layer of photographic film (see electron microscope), or to be detected by a sensor such as a CCD camera. The first practical transmission electron microscope was built by Albert Prebus and James Hillier at the University of Toronto in 1938 using concepts developed earlier by Max Knoll and Ernst Ruska.

Background
In the past, light microscopes were most often used for imaging due to their relative ease of use. However, the maximum resolution that one can image is determined by the wavelength of the photons that are being used to probe the sample. In the early days of microscopy nothing smaller than the wavelength being used could be resolved, whereas nowadays the law of RESOLFT sets the limit for optical microscopes employing such concepts (see microscope). Visible light has wavelengths of 400–700 nanometers, larger than many objects of interest. Ultraviolet could be used, but it soon runs into problems of absorption. Shorter wavelengths, such as X-rays, exhibit a lack of interaction, both in focusing (nothing interacts strongly enough to act as a lens) and in actually interacting with the sample.

Like all matter, electrons have both wave and particle properties (as theorized by Louis-Victor de Broglie), and their wave-like properties mean that a beam of electrons can in some circumstances be made to behave like a beam of radiation. The wavelength is dependent on their energy, and so can be tuned by adjustment of accelerating fields, and can be much smaller than that of light, yet they can still interact with the sample due to their electrical charge. Electrons are generated by a process known as thermionic discharge in the same manner as the cathode in a cathode ray tube, or by field emission; they are then accelerated by an electric field (measured in V/m, or volts per meter) and focused by electrical and magnetic fields (measured in teslas) onto the sample. The electrons can be focused onto the sample providing a resolution far better than is possible with light microscopes, and with improved depth of vision. Details of a sample can be enhanced in light microscopy by the use of stains; similarly with electron microscopy, compounds of heavy metals such as osmium, lead or uranium can be used to selectively deposit heavy atoms in the sample and enhance structural detail, the dense nuclei of the heavy atoms scatter the electrons out of the optical path. The electrons that remain in the beam can be detected using a photographic film, or fluorescent screen among other technologies. So areas where electrons are scattered appear dark on the screen, or on a positive image.

The capabilities of the TEM can be further extended by additional stages and detectors, sometimes incorporated on the same microscope. An electron cryomicroscope is 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.

A TEM can be modified into a scanning transmission electron microscope (STEM) by the addition of a system that rasters the beam across the sample to form the image, combined with suitable detectors.

An analytical TEM is one equipped with detectors that can determine the elemental composition of the specimen by analysing its X-ray spectrum or the energy-loss spectrum of the transmitted electrons.

Modern research TEMs may include aberration correctors, to reduce the amount of distortion in the image, allowing information on features on the scale of 0.1 nm to be obtained (resolutions down to 0.05 nm have been achieved, so far) at magnifications of 50 million times. Monochromators may also be used which reduce the energy spread of the incident electron beam to less than 0.15 eV. Major TEM makers include JEOL, Hitachi High-technologies, FEI Company (from merging with Philips Electron Optics) and Carl Zeiss.

Applications of the TEM
The TEM is used heavily in both material science/metallurgy and the biological sciences. In both cases the specimens must be very thin and able to withstand the high vacuum present inside the instrument.

For biological specimens, the maximum specimen thickness is roughly 1 micrometre. To withstand the instrument vacuum, biological specimens are typically held at liquid nitrogen temperatures after embedding in vitreous ice, or fixated using a negative staining material such as uranyl acetate or by plastic embedding. Typical biological applications include tomographic reconstructions of small cells or thin sections of larger cells and 3-D reconstructions of individual molecules via Single Particle Reconstruction.

In material science/metallurgy the specimens tend to be naturally resistant to vacuum, but must be prepared as a thin foil, or etched so some portion of the specimen is thin enough for the beam to penetrate. Preparation techniques to obtain an electron transparent region include ion beam milling and wedge polishing. The focused ion beam (FIB) is a relatively new technique to prepare thin samples for TEM examination from larger specimens. Because the FIB can be used to micro-machine samples very precisely, it is possible to mill very thin membranes from a specific area of a sample, such as a semiconductor or metal. Materials that have dimensions small enough to be electron transparent, such as powders or nanotubes, can be quickly produced by the deposition of a dilute sample containing the specimen onto support grids. The suspension is normally a volatile solvent, such as ethanol, ensuring that the solvent rapidly evaporates allowing a sample that can be rapidly analysed.

The imaging techniques explained below are particularly important in materials science. Faults in crystals affect both the mechanical and the electronic properties of materials, so understanding how they behave gives a powerful insight. By carefully selecting the orientation of the sample, it is possible not just to determine the position of defects but also to determine the type of defect present. If the sample is orientated so that one particular plane is only slightly tilted away from the strongest diffracting angle (known as the Bragg Angle), any distortion of the crystal plane that locally tilts the plane to the Bragg angle will produce particularly strong contrast variations. However, defects that produce only displacement of atoms that do not tilt the crystal to the Bragg angle (i.e. displacements parallel to the crystal plane) will not produce strong contrast.

Furthermore, the HRTEM technique (see below) allows the direct observation of crystal structure and therefore has an advantage over other methods in that there is no displacement between the location of a defect and the contrast variation caused in the image. However, it is not always possible to interpret the lattice images directly in terms of sample structure or composition. This is because the image is sensitive to a number of factors (specimen thickness and orientation, objective lens defocus, spherical and chromatic aberration), and although quantitative interpretation of the contrast shown in lattice images is possible, it is inherently complicated and may require extensive simulation of the images. Computer modeling of these images has added a new layer of understanding to the study of crystalline materials.

Imaging in the TEM
The contrast in a TEM image is not like the contrast in a light microscope image. A crystalline material interacts with the electron beam mostly by diffraction rather than absorption, although the intensity of the transmitted beam is still affected by the volume and density of the material through which it passes. The intensity of the diffraction depends on the orientation of the planes of atoms in a crystal relative to the electron beam; at certain angles the electron beam is diffracted strongly from the axis of the incoming beam, while at other angles the beam is largely transmitted. 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 below the specimen allow the user to select electrons diffracted in a particular direction.

A high-contrast image can therefore be formed by blocking electrons deflected away from the optical axis of the microscope by placing the aperture to allow only unscattered electrons through. This produces a variation in the electron intensity that reveals information on the crystal structure, and can be viewed on a fluorescent screen, or recorded on photographic film or captured electronically.

This technique (known as Bright Field or Light Field) is particularly sensitive to extended crystal lattice defects in an otherwise ordered crystal, such as dislocations. As the local distortion of the crystal around the defect changes the angle of the crystal plane, the intensity of the scattering will vary around the defect. As the image is formed by the distortion of the crystal planes around the defect, the contrast in these images does not normally coincide exactly with the defect, but is slightly to one side.

It is also possible to produce an image from electrons deflected by a particular crystal plane. By either moving the aperture to the position of the deflected electrons, or tilting the electron beam so that the deflected electrons pass through the centred aperture, an image can be formed of only deflected electrons, known as a Dark Field image.

In the most powerful diffraction contrast TEM instruments, crystal structure can also be investigated by High Resolution Transmission Electron Microscopy (HRTEM), also known as phase contrast imaging as the images are formed due to differences in phase of electron waves scattered through a thin specimen.

Resolution of the HRTEM is limited by spherical and chromatic aberration, but a new generation of aberration correctors has been able to overcome spherical aberration. Software correction of spherical aberration has allowed the production of images with sufficient resolution to show carbon atoms in diamond separated by only 0.89 ångströms (89 pm, one ångström is 0.0000000001 of a meter or 100 picometers) and atoms in silicon at 0.78 ångströms (78 pm) at magnifications of 50 million times. Improved resolution has also allowed the imaging of lighter atoms that scatter electrons less efficiently &mdash; lithium atoms have been imaged in lithium battery materials. The ability to determine the positions of atoms within materials has made the HRTEM an indispensable tool for nanotechnology research and development in many fields, including heterogeneous catalysis and the development of semiconductor devices for electronics and photonics.

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 analysed 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.