Transmission electron microscopy (TEM) is an imaging technique whereby a beam of electrons is focused onto a specimen causing an enlarged version to appear on a fluorescent screen or layer of photographic film (see electron microscope), or can be detected by a CCD camera.
In the past, light microscopes have been used mostly 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; nothing smaller than the wavelength being used can be resolved. Visible light has wavelengths of 400-700 nanometers; larger than many objects of interest. Ultraviolet could be used, but soon runs into problems of absorption. Even shorter wavelengths, such as X-rays, exhibit a lack of interaction: both in focussing (nothing interacts strongly enough to act as a lens) and actually interacting with the sample.
Like all matter, electrons have both wave and particle properties (as demonstrated 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 at the cathode in a cathode ray tube, or by field emission; they are then accelerated by an electric field and focussed by electrical and magnetic fields on to 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 lead or uranium can be used to selectively deposit heavy atoms in the sample and enhance structural detail, the dense electron clouds of the heavy atoms interacting strongly with the electron beam. The electrons can be detected using a photographic film, or fluorescent screen among other technologies.
An additional class of these instruments is the electron cryomicroscope, which includes a specimen stage 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.
In analytical TEMs the elemental composition of the specimen can be determined by analysing its X-ray spectrum or the energy-loss spectrum of the transmitted electrons.
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 vaccuum, biological specimens are typically held at liquid nitrogen temperatures after embedding in vitreous ice, or fixated using a negative staining material such as uraynl 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.
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. If the planes of atoms in a crystal are aligned at certain angles to the electron beam, the beam is transmitted strongly; while at other angles, the beam is diffracted, sending electrons in another direction. In the TEM, the specimen holder allows the user to rotate the specimen to any angle in order to establish the desired diffraction conditions; while an aperture placed below the specimen allows the user to select electrons diffracted in a particular direction. The resulting image shows diffraction contrast, which highlights faults in the crystal structure very clearly. This is very 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.
In the most powerful diffraction contrast TEM instruments, it is possible to produce a diffraction pattern image which is directly analogous to the planes of atoms in the crystal. Although the way contrast arises in these atomic-resolution images is complex, and such images are often interpreted using computer modelling of the electron beam and magnetic lenses, these images have added a new layer of understanding to the study of crystalline materials.