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Scanning Electron Microscopy

Scanning Electron Microscopy

The scanning electron microscope (SEM) is an important tool in modern forensic science due to its wide range of applications. SEM allows the rapid analysis of elements that compose very small specimens and the conclusive determination of the origin of many materials that are crucial to the chain of evidence . Paint particles, fibers (both natural and artificial), bullet fingerprints, gunshot residue , counterfeit bank notes, forged documents, and trace evidence are all examples of specimens that can be analyzed using the scanning electron microscope. Scanning electron microscopy also renders detailed three-dimensional (3-D) images of extremely small microorganisms, 3-D anatomical pictures of insect, worm, spore, or other organic structures, and the analysis of gems and gem fragments.

Conventional microscopes use light and several lenses to magnify images, whereas SEM uses electron beams to sweep the surface of specimens, producing magnified images in black and white. In most SEMs, samples are placed in a vacuum chamber after being adequately prepared to conduct electricity. Once the sample is in the chamber, the air is extracted and an electron gun at the top of the chamber emits a beam of electrons, which passes through a series of magnetic lenses that condense the beam into an extremely fine focus, capable of sweeping nano spots on the sample surface. A scanning device near the bottom of the vacuum chamber controls the movement of the electron beam across the specimen, row by row. As the electron beam sweeps the surface, it excites electrons present in the atomic structure of molecules, causing some of them to escape from the surface. These escaping electrons, known as deflected secondary electrons, have specific energies that can be measured. As they are released from each area of the sample, they are collected and counted by a detector that sends their amplified signals. The various electronic energies produced are analyzed by computer software, and the resulting image is displayed on a computer monitor.

Some modern SEMs offer an additional advantage for forensic purposes because of new methods of biological sample analysis that do not corrupt the specimen, a major drawback with conventional SEMs. In conventional electron microscopy, biological samples have to be dehydrated and then coated with a material that conducts electricity, such as a thin layer of gold or carbon. Modern SEMs allow the adjustment of the internal pressure in the chamber to dissipate the electric charge that would otherwise charge the sample, thus dispensing with coating and dehydration. Examples of non-conductive materials that require special preparation in conventional SEMs are paper, paint, textiles, bone, hair, and glass .

Each chemical element consists of an atomic structure composed by a given number of particles in the nucleus and of electrons vibrating in different levels or shells around the nucleus, each at a specific distance from the nucleus. Electrons in different shells ("orbits") have different energies and the atomic weight of the nucleus determines the quantity of electrons of an atom. Atoms are usually neutral because all their positive and negative particles are in a state of dynamic electrical balance. However, when free atoms collide or when they are bound through molecular chemical reactions, some atoms gain or lose an extra electron, thus becoming positive or negatively charged (cations or anions). When the electron beam of a SEM hits the sample, it deflects two types of electrons from the sample: inelastic electrons and elastic electrons. Inelastic electrons are low-energy particles that give information about the topographic variations on the sample surface and are responsible for 3-D black and white images. They are also known as secondary electrons and most of them have charges inferior to ten electron Volts (<10 eV). Elastic electrons are those that collide with the electrons generated by the SEM (that are present in the beam). The collision of electrons produces specific energy quanta that are retained by the elastic electrons. By calibrating the microscope to different beam intensities, analysts can study several types of data provided by elastic electrons, such as the sample composition and crystallographic structure of the surface, the internal structure of semi-conductor materials, the distribution and energy levels of phosphorous compounds, and information about the elements and chemicals present in the several layers of the surface.

Forensic analytical tests such as scanning electron microscopy, spectrometers, chromatography , and x-ray dispersion aim at producing individualized evidence that allows the identification and origin of samples and the accurate interpretation of data in relation to a crime or a suspect investigation or to help explain an explosion, arson , or airplane crash. Modern scanning electron microscopy provides nondestructive analysis of both organic and inorganic samples. Another application of this method in forensics is the analysis and identification of dust particles in the air of indoor environments to either assess the air quality or to detect possible pathogens (disease-causing organisms) or hazardous substances. Mineral grains (such as carbonates, glass, quartz, or mica), biological materials (such as mold spores , pathogen spores, insect particles, skin cells, and rodent fecal dust), fibers (such as hair, textile fibers, carpet fibers, cellulose, and asbestos), and miscellaneous particles (such as metallic particles, paint, soot, rubber, and plastic) are all materials that have been used in forensic analysis done with scanning electron microscopy.

see also Accelerant; Aircraft accident investigations; Analytical instrumentation; Arson; Artificial fibers; Ballistic fingerprints; Bomb (explosion) investigations; Document forgery; Fibers; Filaments; Hair analysis; Handwriting analysis; Ink analysis; Isotopic analysis; Minerals; Organic compounds; Paint analysis; Point-by-point analysis.

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Electron Microscopy

Electron Microscopy

The light microscope (LM) is limited in its resolution to about 0.25 micrometers. If two objects are closer together than that, they blur together and cannot be distinguished by the LM. The electron microscope (EM) overcomes this limitation and achieves resolutions down to 0.2 nanometers , allowing useful magnifications of biological material up to several hundred thousand times, and even more for nonbiological specimens. The EM achieves this by using a beam of electrons instead of visible light. Resolution is governed by the wavelength of illumination, and an electron beam has a much shorter wavelength (about 0.005 nanometers) than visible light (about 400 to 750 nanometers). Electron microscopes can therefore resolve objects as small as individual protein and deoxyribonucleic acid (DNA) molecules and pores in cell membranes.

The electron beam of an EM is generated by a heated tungsten wire (cathode) and accelerated down an evacuated column by a charge difference of typically 60,000 to 100,000 volts between the cathode and a grounded, mushroom-shaped anode. After passing through a hole in the center of the anode, it is focused on the specimen by electromagnets, which take the place of the glass lenses of a light microscope.

The Transmission Electron Microscope

In the transmission electron microscope (TEM), the electron beam passes through ultrathin tissue sections or small specimens, such as viruses. After passing through the specimen, the electrons strike a fluorescent screen and produce an image. The image can also be captured on photographic film or with a camera that digitizes it for storage on a computer.

Specimens for the TEM are typically fixed with aldehyde and stained with heavy metals, such as osmium, that will absorb or scatter electrons. The specimen is then dehydrated and embedded in a plastic resin. When it hardens, the resin is cut into sections 60 to 90 nanometers thick with a glass or diamond knife. Very tiny particles such as viruses and purified cell organelles can be viewed without sectioning by depositing them on a thin membrane. This membrane is treated with a heavy metal "negative stain" so that the specimen stands out as a light image against a dark background.

Areas of a specimen that bind the most osmium absorb the most energy from an electron beam, and are called electron-dense regions. Areas that bind less of the stain allow electrons to pass through more freely and are described as electron-lucent regions. Electrons that pass through the lightly stained, electron-lucent regions lose relatively little energy and produce relatively bright spots of light when they strike the screen. The more heavily stained, electron-dense regions cause some electrons to lose energy and others to be deflected from the beam, and thus produce dimmer spots on the screen. TEM images are essentially shadows caused by accumulations of the heavy metal on cellular structures or, in the case of negative staining, on the supporting membrane.

The Scanning Electron Microscope

The scanning electron microscope (SEM) is used to examine a specimen coated with vaporized metal ions (usually gold or palladium). An electron beam sweeps across the specimen surface and discharges secondary electrons from the metal coating. These electrons produce an image on a monitor similar to a television screen. The image on the monitor can be photographed or recorded with a digital camera. The SEM cannot see through a specimen as the TEM does, but can see only the surface where the metal coating is.

The SEM is capable of less resolution and useful magnification than the TEM. However, it produces dramatic three-dimensional images that can yield more information about surface topography than the flat images usually produced by TEM.

Other Variations in Electron Microscopy

Both SEMs and TEMs can be equipped with a detector that monitors X rays given off by a specimen when it is bombarded by electrons. Other types of microscopes irradiate the specimen with ions or X rays and record ions, electrons, or X rays given off by the specimen. In both cases, the emitted particles and radiation yield information about the chemical composition of the specimen.

A scanning tunneling microscope measures the vertical movement of a tiny probe that is dragged over a specimen, producing a line representation of that movement. An atomic force microscope operates on a similar principle, but measures forces of attraction and repulsion between the specimen and the probe as the probe moves across the surface. In either case, multiple scan lines side by side produce images of the specimen surface, revealing details as small as the "atomic terrain" of individual molecules.

see also Light Microscopy; Microscopist

Sara E. Miller and Kenneth S. Saladin

Bibliography

Berger, Dee. Journeys in Microspace: The Art of the Scanning Electron Microscope. New York: Columbia University Press, 1995.

Gilmore, C. P. The Scanning Electron Microscope: World of the Infinitely Small. Greenwich, NY: Graphic Society, 1972.

Microworld Internet Guide to Microscopy. <mwrn.com/guide/electron_microscopy/microscope.htm>. Includes lecture notes and guides to EM techniques and instrumentation.

Slayter, Elizabeth M., and Henry S. Slayter. Light and Electron Microscopy. New York: Cambridge University Press, 1992.

WWW Virtual Library: Microscopy. <http://www.ou.edu/research/electron/mirror/>. Numerous links to other sites on all aspects of microscopy.

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electron microscope

electron microscope A form of microscope that uses a beam of electrons instead of a beam of light (as in the optical microscope) to form a large image of a very small object, such as a cell organelle, a virus, or a DNA molecule. In optical microscopes the resolution is limited by the wavelength of the light. High-energy electrons, however, can be associated with a considerably shorter wavelength than light; for example, electrons accelerated to an energy of 105 electronvolts have a wavelength of 0.04 nanometre, enabling a resolution of 0.2–0.5 nm to be achieved. The transmission electron microscope (see illustration) has an electron beam, sharply focused by electron lenses (coils producing a magnetic field or electrodes between which an electric field is created), passing through a very thin metallized specimen (less than 50 nanometres thick) onto a fluorescent screen, where a visual image is formed. This image can be photographed. The scanning electron microscope can be used with thicker specimens and forms a perspective image, although the resolution and magnification are lower. It is used particularly for examining surface features of small objects, such as pollen grains. In this type of instrument a beam of primary electrons scans the specimen and those that are reflected, together with any secondary electrons emitted, are collected. This current is used to modulate a separate electron beam in a TV monitor, which scans the screen at the same frequency, consequently building up a picture of the specimen. The resolution is limited to about 10–20 nm. See also field-emission microscope; field-ionization microscope.

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electron microscope

electron microscope Microscope used for producing an image of a minute object. It ‘illuminates’ the object with a stream of electrons, and the ‘lenses’ consist of magnets that focus the electron beam. Smaller objects can be seen because electrons have shorter wavelengths than light, and thus provide greater resolution. The image is obtained by converting the pattern (made by electrons passing through the object) into a video display, which may be photographed. These microscopes can magnify from 2000 to a million times.

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electron microscope

electron microscope n. a microscope that uses a beam of electrons as a radiation source for viewing the specimen. The resolving power (ability to register fine detail) is a thousand times greater than that of an ordinary light microscope.

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electron microscope

e·lec·tron mi·cro·scope • n. Physics a microscope with high magnification and resolution, employing electron beams in place of light and using electron lenses.

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electron microscope

electron microscope: see microscope.

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