Scanning Electron Microscope Specimens

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Scanning Electron Microscope

A scanning electron microscope (SEM) is of course a microscope, which means it is used to observe and analyze specimens on an extremely small scale that would not otherwise be visible to the naked eye. The primary difference between this microscope and a typical optical lab microscope is how we look at the specimens. Optical microscopes use light to magnify the objects to the objects in question. Light hits the object and light waves bounce back. They are magnified through the series of lenses, enlarging it from the actual size. The problem with light waves is that there is a limit to how small we can see them. Light waves can only resolve detail to a certain limit of 0.2mm and magnify effectively to 1000X.

This is where a scanning electron microscope comes into play to get past the viewing obstacle of the optical light microscopes. SEM images are not produced by light, but by means of low energy secondary electrons emitted from the surface of a specimen when a very narrow beam of high energy primary electrons strikes it. Electrons have wavelengths associated with them depending on their energy. These wavelengths can be resolved to much finer details than optical microscopes. So how does an SEM work? First electrons are ejected off a fine filament running at a high voltage (10V-30V), and then they travel down a “gun” where various magnetic lenses condense them into a narrower beam. Finally, they are focused toward a single spot which is the specimen.

The electrons then interact with the specimen's electrons. Hitting the specimen generally results in two things, backscatter and creating secondary electrons. Backscatter electrons are originally from the beam. They come in and essentially bounce off the electrons around the individual atoms of the specimen. Backscatter electrons occur more often as the atomic number of the specimen increases.

Secondary electrons occur when the beam electrons transfer some of the energy to the electrons of the specimen atoms. Electrons in the specimen become excited and they will eject out. Topography of the specimen tends to be emphasized in the secondary images. Electrons are then gathered by various detectors (backscatter and secondary detectors). An image is made depending on how many electrons of a particular type are ejected from a certain spot. More electrons create a bright spot, while a few ejected electrons create a dark spot.

The microscope itself consists of a) a source of primary electrons that is somewhat like light bulb of filament. The electrons are accelerated towards the specimen by a voltage. B) A series of magnetic lenses that gathers the electrons into a very narrow pencil like beam that is about 1/10,000mm in diameter. c) A means of causing this beam to scan the specimen. d) An electron detecting and display system. The image from the microscope is displayed on a screen.

When the high energy primary electrons penetrate the specimen surface and pass deep into the specimen, they knock out large numbers of low energy ionization electrons, losing their own energy in the process. The low energy electrons cannot travel far within the specimen without being recaptured, so that only those produced vary close to the specimen's surface can escape. As the image is a result of these low energy electrons, only the surface of the specimen is seen. Furthermore, as the primary electron beam is extremely narrow and the secondary electrons do not have to be focused, but merely collected for display, a very large depth of field is achieved which is some five hundred times greater than that of an optical light microscope at the same magnification.

Samples that are electrically conducting can be readily examined directly, but non-conducting ones such as biological samples, have first to be given an electrically conducting surface by coating them with a very thin of metal, usually gold or a mixture of gold and palladium. Their structure can also be preserved either by incubation of specimen in solution in a fixative such as glutaraldehyde or formalin. This preparation, together with the fact that the whole microscope works under vacuum, means that in general only non-living specimens can be studied under the microscope. The range of useful magnification is form about fifteen to 20,000 times on biological specimens and somewhat more on other material, such as metals which do not need coating.

In scanning electron microscopy the very narrow electron beam is made to scan the specimen in a screen, similar to how a television set works. The times it takes to scan a sample when recording an image is generally quite long, about forty to hundred-fifty seconds. This relatively slow process of building up a picture of the specimen is necessary because the amount of information or detail present in a final picture is approximately proportional to the length of time the primary beam stays in any one place on the specimen.

The final picture of the sample is three dimensional in appearance because of the effect of perspective and of the presentation in light and shade on the screen. The light and shade appear because for surfaces sloping towards the electron detector a large number of electrons are collected and the image on the screen appears bright; for surfaces that slope away from the detector there are fewer collected and so these surfaces appear darker. This can be compared to photographing an object under strong side lighting, usually used in photography to produce a aesthetic visual appearance, as well as enhancing the feeling of a two dimensional appearance. This leads to arguably the most valuable feature of the scanning electron microscope, the ease of interpreting images on the screen. An image looks right even to the untrained person operating the instrument.

In general, scanning electron microscopes are as controllable as conventional photography: an object can be positioned in any way, magnification can be chosen either in steps or by zooming and the range of image brightness can be controlled. The depth of field can also be exchanged for better resolution. Manipulation of these features enables a considerable amount of control over the appearance of an image. The natural micro world is full of three dimensional forms that many find aesthetically pleasing and at present it is only be means of this technique or method that scientists can tap into it.

Scanning electron microscopy has a wide variety of applications to the world today. Over the past twenty years forensic scientist have use SEM for various imaging and analytical microscopy applications. Using the superior imaging capability to produce high magnification and high resolution electron micrographs, scientists have been able to detect, characterize and identify otherwise unseen valuable microscopic clues to help solve criminal cases. In the last ten years, SEM technology has grown exponentially with the introduction of environmental low vacuum pressure. With this advance, forensic scientists are able to examine virtually any material on a microscopic level and in some cases approaching molecular scales.

Crime laboratories worldwide that can afford to purchase a SEM, most often use to search for primer gunshot residue on tape lifts obtained from the alleged shooter hands or clothing. Gunshot residue produced from the primer components of the fired cartridge, typically have a spherical shape and contain the elements lead (Pb), barium (Ba) and antimony (Sb). The SEM can check if the residue matches the gunshot residue.Comparison of materials is also a common use for the SEM. Often subject materials such as chips of glass, paint and metals are characterized in the SEM and then comparisons are made to known materials collected form the objects that are suspected to be the source of the chips. For example, if a car struck a painted wooden wall and the driver fled the scene and the police captured the suspect and impounded the car a few days later. Small paint chips were then found on the car in a damaged area corresponding to the height of the damage to the wall. Paint chips can then be removed from the car and the wall for comparison analysis using the scanning electron microscope.

Cross sections of the paint chips will reveal a multi layered structure and each layer examined in the SEM to measure film thicknesses of each paint layer and to produce the elemental profile of each layer. Both sets of paint chips will appear to be identical thus proving incontrovertibly that the suspected driver hit the painted wall.

We can see that the SEM is a very powerful tool in the scientific world, making a great impact with its wide range of applications.