Electron microscopy: transmission EM

Principle

Transmission electron microscopy (TEM) is an imaging technique whereby a beam of electrons is transmitted through a specimen and next an image is formed (see Electron microscopy). Fig. 1 compares the basic pathways of radiation in optical microscopy and in TEM.

Fig. 1 Comparison of optical microscopy and in TEM.

In the early days of microscopy nothing smaller than the wavelength being used could be resolved, whereas nowadays subdiffraction techniques (see Optical microscopy: super-resolution techniques) and the law of RESOLFT (REversible Saturable Optical Fluorescence Transitions) sets the limit for optical microscopy. (The RESOLFT concept increases the classical resolution (see Optical microscopy) by raising the beam intensity.) The wave-particle nature of all matter allows considering the electron beam as a beam of radiation. Its wavelength is dependent on the kinetic energy (equations 3-6 of Electron diffraction), and so can be tuned by adjustment of accelerating fields. It 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 emission by a cathode or by field emission (a form of quantum tunneling in which electrons pass through a barrier in the presence of a high electric field.) Thermionic emission is the flow of charge carriers (electrons or ions), from a surface or over some other kind of electrical potential barrier, caused by thermal vibrational energy overcoming the electrostatic forces restraining the charge carriers. (A hot metal cathode emitting into a vacuum is the classical example (the Edison effect). After acceleration the electrons are focused by electrical and magnetic fields (lenses) onto the sample. Contrast can be enhanced by “staining” the sample heavy metals such as Os, Pb, U. The dense nuclei of the heavy atoms scatter the electrons out of the optical path. The areas where electrons are scattered appear dark on the screen and the electrons that remain generate the image.

Resolution of the TEM is limited primarily by spherical aberration (see Light: the ideal lens), but a new generation of aberration correctors reduce the amount of distortion in the image and consequently this limitation.

A monochromator may also be used which reduce the energy spread of the incident electron beam to less than 0.15 eV. (A monochromator transmits a narrow band of wavelengths of radiation chosen from a wider range of wavelengths available at the input.)

High Resolution TEM

In the most powerful diffraction contrast TEM instruments equipped with aberration correctors, crystal structure can also be investigated by High Resolution TEM (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.

Software correction of spherical aberration for the HRTEM has allowed the production of images with sufficient resolution to show carbon atoms in diamond separated by only 0.089 nm. The ability to determine the positions of atoms within materials has made the HRTEM an important tool for nano-technologies research, metallurgic studies and semiconductor development.

TEM can be modified into scanning TEM (STEM) by the addition of a system that rasters the beam across the sample to form the image, combined with suitable detectors. The rastering makes these microscopes suitable for mapping by energy dispersive X-ray spectroscopy, electron energy loss spectroscopy (EELS) and annular dark-field imaging. These signals can be obtained simultaneously, allowing direct correlation of image and quantitative data.

By using a STEM and a high-angle detector, it is possible to form atomic resolution images where the contrast is directly related to the atomic number. This is in contrast to the conventional HRTEM, which uses phase-contrast, and therefore produces results which need interpretation by simulation.

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

Application

TEM is used heavily in both material science/metallurgy and biological sciences. For biological specimens, the maximum thickness is roughly 1 μm, but ideally some 0.1 μm. To withstand the instrument vacuum, biological specimens are typically held at liquid N₂ 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 cell reconstructions and 3D reconstructions of individual molecules via Single Particle Reconstruction. This is a technique in which large numbers of images (10,000 - 1,000,000) of ostensibly identical individual molecules or macromolecular assemblies are combined to produce a 3D reconstruction. As molecules/assemblies become larger, it becomes more difficult to prepare crystals for high resolution. For single particle reconstruction, the opposite is true. Larger objects actually improve the resolution, nowadays, also with aid of computational capabilities down to 0.5 nm.

More Info

There are a specific number of techniques to prepare samples used in material science (e.g. crystallography) and geology. They will not be discussed here.

The contrast in a TEM image is not like the contrast in a light microscope image. A crystalline material (e.g. proteins) 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. This technique is known as Bright Field or Light Field.

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.

There are a number of drawbacks to the TEM technique:

- sample preparation is time consuming;

- sample preparation has the risk of introducing artifacts;

- there is a risk that the sample may be damaged by the electron beam;

- the small field of view, which raises the possibility that the region analyzed may not be characteristic of the whole sample.