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Electron Microscopy - Introduction, The Transmission Electron Microscope (TEM), The Scanning Electron Microscope (SEM), Elemental Identification in

electrons images surface specimen

DOUGAL G. McCULLOCH
RMIT University

Introduction

Electron microscopes are scientific instruments that use beams of energetic electrons to examine objects on a very fine scale. Based on the design of optical microscopes, electron microscopes exploit the fact that fast moving electrons have a much smaller wavelength than visible light, which results in high-resolution images. Electron microscopes can routinely image at magnifications over 1,000,000×, compared to light microscopes which are limited to magnifications of the order of 2000×.

Electron microscopes use an electron gun to generate the beam of energetic electrons. Whereas the light microscope uses glass lenses to magnify and focus images, the electron microscope uses magnetic lenses to magnify and focus images. Since electrons cannot travel freely in air, electron microscopes are built into airtight metal tubes or “columns” and use vacuum pumps to remove all the air from within the microscope.

There are two main types of electron microscope: (1) the transmission electron microscope (TEM) and (2) the scanning electron microscope (SEM). Figure 28 compares images of a dinoflagellate (Gymnodinium), which is a type of unicellular algae, taken using (a) an ordinary light microscope, (b) a TEM, and © a SEM. In general, the TEM image provides high-resolution information on the internal structure of a specimen while the SEM provides a detailed image of the surface structure of a sample. It is clear that both electron microscopes provide higher resolution images than is possible using a light microscope.

The Transmission Electron Microscope (TEM)

The basic layout of a TEM is shown in Figure 29. It consists of an electron gun as the source of “illumination” at the top of a column containing a series of lenses, the specimen, and the imaging system. The layout of the TEM closely resembles that of a compound light microscope, which is also shown alongside for comparison purposes. Both microscopes use a condenser lens to focus the illuminating beam (either electrons or light) onto the sample and an objective lens to produce a focused and magnified image of the illuminated area. One or more projector lenses are then used to project a magnified image of the specimen onto the imaging system. In the case of an optical microscope this is the eye, film, or a charge-coupled device (CCD), while in a TEM images are viewed on a screen that fluoresces when struck by electrons. Traditionally, grayscale still images were captured on silver halide film or plates situated beneath the viewing screen that were exposed to the electron image and then processed to a negative, as in conventional photography. Photographic capture of images is gradually being replaced with digital capture using electron-sensitive CCD cameras.

Two factors complicate the imaging of samples in a TEM. First, due to the limited penetration of electrons in matter, specimens for TEM must be extremely thin (approximately 0.1 micron). Second, since the specimens must be inserted into the electron microscope column which is under vacuum, they must be completely dry. This latter point requires biological samples to be dehydrated and fixed prior to viewing. There is a range of TEM specimen preparation techniques available depending on the type of sample. Solid, inorganic samples can be thinned using a combination of mechanical, chemical, and ion beam thinning. Thin biological specimens are normally prepared using ultramicrotomy, which involves slicing thin sections of the sample using a sharp glass or diamond knife. Surfaces can be replicated as thin films of carbon and shadowed with heavy metals to produce remarkably realistic images of surface relief. As with most kinds of microscopy, specimen preparation is an important and specialized area of expertise.

Contrast in TEM images can arise in several ways. Variation of mass and/or thickness gives rise to contrast due to greater absorption or scattering of electrons from heavier and/or thicker parts of the specimen. To increase contrast in biological specimens, tissues are often “stained” with heavy metals. Another contrast mechanism is known as phase contrast. Unlike phase contrast in optical microscopy, phase contrast results from the interference between electrons, which have different phases after passing through the specimen. Phase contrast can be used to directly image the crystal structure of a specimen at atomic resolution.

Electrons passing through a specimen can be “reflected” off planes of atoms in a process called diffraction. In addition to producing images, a TEM can be configured to produce electron diffraction patterns from specimens. Similar to X-ray crystallography, electron diffraction can be used to determine the structure of a crystalline specimen.

The Scanning Electron Microscope (SEM)

The layout of the SEM is very different from a TEM, as shown schematically in Figure 30. The SEM column consists of the electron gun and then several magnetic lenses, which are used to focus the electron beam into a small spot onto the sample surface. Unlike the TEM, there are no lenses after the specimen in a SEM. Scan coils are used to deflect the finely focused electron beam to create a tiny rectangular grid of parallel lines (a raster, as in a cathode ray tube) over the area of interest, while detectors measure the number of electrons that are displaced from each point on the surface. An image of the scanned area is generated on a video display, which is synchronized with the scan coils of the SEM so that the relative position of features is correctly displayed. The magnification of an SEM image is governed by the difference between the dimensions of the video display (normally fixed) and that of the area scanned on the specimen surface. Increasing magnification is achieved by scanning smaller and smaller areas on the specimen. Grayscale images are captured and stored digitally. The resolution of an SEM is determined primarily by the size of the electron spot, which is of the order of 1 to 2nm on modern instruments.

Specimens for conventional SEM need to be electrically conducting so that charge built up in the surface from the incident electron beam can be conducted away. This problem can be overcome by insulating specimens with a covering of thin, electrically conducting coatings, typically of gold or platinum. Conventional SEMs are also limited by the fact that a high vacuum must be maintained in the sample environment, requiring completely dry specimens. Low vacuum or “environmental” SEMs can overcome both these limitations. These
instruments use pressure-limiting apertures to maintain the gun and column at high vacuum while allowing much higher pressures in the specimen area. Therefore hydrated specimens can be imaged in their natural state. In addition, specimen charging is neutralized as positive ions are attracted to the surface from the surrounding gas, so non-conducting specimens can be imaged without the need for coatings.

There are two main types of electron detectors used in an SEM. The first measures the number of electrons, which have backscattered off the sample surface (known as backscattered electrons). The second detector measures the number of electron, which are ejected from the material (known as secondary electrons) following collisions with the incident electron beam. Images generated using secondary electrons are extremely sensitive to surface morphology, while those generated using backscattered electrons are sensitive to the atomic weight of different regions of the sample. In both kinds of microscopes, it is relatively easy to make stereo pairs of images by tilting the specimen a few degrees along an axis parallel to the viewing direction. These often give a much better impression of the surface relief than single pictures.

Although images derived from collected electrons in an SEM are grayscale, color images can be artificially generated by combining secondary and backscattered images collected from the same sample. An example of a colorized SEM image of an insect is shown in Figure 31.

Elemental Identification in Electron Microscopy

When an electron beam interacts with matter, X-rays and other electromagnetic radiation can be emitted as a result of collisions between the incident electrons and electrons within the atoms that make up the specimen. The energies of the
emitted X-rays are characteristic of the element from which they originated. It is thus possible to perform elemental analysis on minute specimens and samples in an electron microscope by measuring the energies of these X-rays. Modern electron microscopes also allow the spatial distribution of elements in a sample to be determined in a technique known as elemental mapping. Some materials such as CRT phosphors, minerals, and some organic compounds emit light when bombarded with electrons, and “cathodoluminescent” images can be made using this light in the SEM. By using three exposures and color filters, true-color images of the luminescence can be reconstructed that have a useful diagnostic value.

Complimentary Techniques

Another technique, which does not involve electron optics or beams, can be used to provide high magnification images of specimens. This is scanning probe microscopy (SPM), where an atomically sharp, needle-like tip (called a probe) is scanned across the surface to be studied. As this tip is scanned, variations in the topography of the surface are measured. An atomic-scale image of the surface can be built up by rastering the probe over an area of the surface. The two main variants of SPM are scanning tunneling microscopy (STM) and atomic force microscopy (AFM). STM uses a small electrical current that can pass between the tip and the surface of a conducting sample to monitor variations in surface topography. In AFM, variations in the surface are detected via the inter-atomic force between atoms on the tip and those on the surface of either an insulating or conducting sample.

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over 6 years ago

I don't suppose one could get information on the original source, or possibly see the figures mentioned within the text? Thanks!