Scanning electron microscopes (SEM) provide forensic investigators with critical imaging and analytical capabilities that are not available from other techniques. In particular, they can resolve features as small as a nanometer, allowing useful magnifications of 100,000 times or more.

Even at very high magnifications, scanning electron microscopes have large depth of field, allowing them to capture well-focused images of complex three-dimensional objects. In conjunction with X-ray microanalysis, they can determine the elemental composition of the samples as small as a few micrometers or map the distribution of elements in larger samples with micrometer spatial resolution. They are widely used for the analysis of gunshot residue; tool mark investigations; ink and paper analysis; paint, hair, and fiber matching; and the identification of other trace evidence, such as pollen and diatoms, that can link a suspect to a crime scene.

Unlike optical microscopes, which form a real image from transmitted or reflected light, a scanning electron microscope forms a virtual image on an electronic display. A finely-focused beam of electrons scans the sample surface in a raster pattern and the brightness of each point in the displayed image represents the strength of the imaged signal generated at the corresponding point in the sample when it is illuminated by the beam.

The imaged signals are created by interactions between beam electrons and sample atoms as the beam penetrates into the sample. The depth of penetration is determined by the accelerating voltage of the beam and the density of the sample, and ranges from a few nanometers to a few micrometers. SEM is generally regarded as a surface imaging technique. Different interactions yield signals, each with different characteristics. The most common signals include: secondary electrons (SE), used primarily for high resolution and high topographic contrast; backscattered electrons (BSE), used for material contrast (atomic number); and characteristic X-rays, used to determine elemental distribution and composition.

Topographic detail for tool mark analysis.

backscattered elctron images show strong material contrast.

In general, the sample must be clean, dry, non-volatile, and electrically conductive. The sample must tolerate high vacuum and not interfere with the operation of the vacuum system. Due to the electron bombardment, charge will build up on the sample surface. If the sample is conductive, current flowing from the grounded sample stage neutralizes the charge. Non-conductive samples are often coated with a conductive material to provide a path to ground. Most SEM sample preparation focuses on meeting these requirements and samples that meet them in their native state can generally be introduced directly into the sample chamber.

Barium X-ray map overlaid on a BSE image.

Gunshot residue particle.

Two issues are especially important for forensic SEM applications: preservation of the sample’s integrity and evidentiary value and validation of the imaging and analytical results. Preserving the sample’s value as evidence requires that it not be modified by the examination so that it can be reexamined in its original condition by another party or at another time. In this context, a specialized kind of SEM with greatly relaxed vacuum requirements can be very valuable like environmental (ESEM) and low vacuum (LVSEM). Although the details of their operational mechanisms differ, they relax the sample requirements to some degree, ideally enough to permit examination without any preparation that would alter the sample.

Validation refers to the need to demonstrate in court that the microscope was calibrated and working properly and the methods used conform to generally accepted standards. Special validation software is designed to automatically check several SEM functions, like column alignment, Energy Dispersive Xray Spectroscopy (EDS) and BSD detector efficiency, and EDS imaging calibration in respect to stage movements. A sample mount containing several certified standards, like a tin balls sample, a SIRA grid, and a special stub that allows fully automatic SEM/EDS diagnosis. It also includes a certified particle test sample that would test for field stitching errors and EDS classification. Special GSR standards are also available, designed for the adjustment, calibration, and validation of analytical SEM/EDX systems when used for automatic analysis of GSR particles. All samples are certified and each one serialized, fully characterized and documented.

Forensic applications of SEM include analysis of gunshot residue, shooting distance, tool marks, ink, paper, fiber, hair, paint coatings, and many more. Scanning electron microscopy (SEM) has become an indispensable tool for forensic investigation, shedding light microscopic traces of evidence through imaging, image comparison, and X-ray microanalysis. Forensic samples can stem from a wide range of micro-materials. Here we describe four applications that demonstrate its power in forensic applications.

Copper threads inside plastic wire, magnified.

Backscattered electron image of metal filament and glass particle.

Gunshot Residue
In forensic sciences, gunshot residue analysis (GSR) is an important issue. The analysis of gunshot residue can be done with an SEM linked to an X-ray microanalysis system equipped with a GSR analysis software package. This software package allows automated classification of GSR particles (typically containing Pb, Sb, and Ba) and other indicative particles such as those which originate from the bullet and contain a large amount of lead (Pb). Other elements of interest are titanium (Ti) and zinc (Zn) which are found in lead-free ammunition. Environmental related particles can be characterized by the system to provide additional information about the suspect or crime scene.

Particles are generally collected from the suspect onto small stubs by using a tape lift method. Each individual stub is fully scanned, classifying all measured particles during an unattended (overnight) run. The search for particles can go as small as 0.3 microns in diameter. Once classified as potential gunshot residue particles, these particles are revisited in order to perform a manual confirmation.

Multiple SEM images stiched together.

Shooting Distance
During the investigation of a shooting, there are usually several different perceptions of what happened. Elements such as the struggle over a firearm or where the victim and shooter were standing in relationship to one another are questions that need unbiased answers.

Firearm examiners routinely examine a shooting victim’s clothing for bullet holes and other evidence that may determine the distance from the muzzle of the firearm to the clothing. Determinations are made as to which holes are bullet entrance holes and which are bullet exit holes. The area around bullet entry holes are examined for patterns of gunshot residues. If residue patterns are detected, then these are compared with test targets produced by firing the firearm in question at various known distances which allows the examiner to approximate the distance from the object that was shot to the muzzle end of the firearm.

Shooting distance investigations are normally carried out using wet chemicals. In using these chemicals, a coloring will appear if firearm discharge residue (FDR) particles are present, such as gunshot residue (GSR). Often, however, such tests are destructive so the evidence is no longer available for cross examination.

By using the SEM’s low vacuum mode, the uncoated sample can be easily investigated for bullet wipe or patterns around the bullet hole. A special software routine scans the sample for particles, stores all images, and even performs real GSR analysis. By scanning in cross-mode, within minutes an overview is made of all present particles. The indicated distribution helps to determine the shooting distance.

Tool Marks
A tool mark is considered to be any impression, cut, or abrasion caused by a tool coming into contact with another object. Most often, tool marks are encountered at burglary scenes that involve forcible entry into a building, but they are found in homicide cases where an axe or knife strikes bone. Generally these marks occur in the form of indented impressions into a softer surface or as abrasion marks caused by the tool cutting or sliding against another object.

Just as firearms possess and leave individual characteristics from their manufacture and use, so do tools such as pry bars, axes, and knives. They leave marks that can be used to positively identify the use of a particular tool. Tool mark examinations are comparative examinations, whereby a tool mark or a tool mark cast is compared to known tool marks produced in the laboratory by the suspect tools. The known and unknown marks are compared microscopically using a comparison microscope. Traditionally, examination is carried out using an optical microscope. Due to the lack of focus depth, optical microscopes do not have the ability to reveal fine detail at higher magnification. As many tools do not have a flat surface, examination under an optical microscope is challenging.

The SEM has a far superior depth of field so it can produce images covering a wide range of magnifications and depths on many types of surfaces, including rough and curved surfaces. The sample can be easily rotated over 360°. A special holder (ballistic substage) helps to rotate two objects at the same time.

Trace Evidence
The evaluation of a vehicle’s lighting condition at the time of an accident is an important task that is accomplished by examining the lamp and lamp-remains. In traffic accidents at night, it is important to establish whether headlamps and rear lights were on or off at the moment of impact. Daytime accidents can bring up questions as to whether the turn signal lamps of a car were activated.

The effect of impact on bulbs is dependent on the status of the filament (hot, cold) and whether or not the filament envelope was broken. In case of a broken envelope at the time of impact in combination with a hot filament, oxidation of the wire often takes place. Also, small spherical glass particles can be found on the filament. On hitting the hot filament, glass splinters will melt and form perfect spheres as the molten glass does not wet the surface of the wire.

Using backscatter imaging, one can easily recognize the small particles in comparison to the tungsten wire. Using the EDX microanalysis in spot mode will show the chemical composition of the small particles within seconds. As oxidized wires are not electrically conductive, a metal coating is normally required on the sample. By using low vacuum technology it is possible to investigate these types of samples without any sample preparation.

The ability of SEM to provide both high resolution imaging and compositional analysis makes it an excellent tool for forensic investigations. It is already well established for applications such as the analysis of gunshot residue, shooting distance, tool marks, and trace evidence.

Hans Kruesemann is responsible for the forensic SEM marketing and applications, for FEI Company. He started his career at the Philips Research Laboratory, Eindhoven, The Netherlands, and via the Central Analytical Laboratory Lighting of Philips, as chemist. In 1994, Kruesemann started at Philips/FEI Electron Optics (now FEI Company) and since1996, Kruesemann has been involved in forensic applications, working closely with Eastern Analytical on the development of Gunshot Residue (GSR) Particles Analysis Software and other related forensic products. He can be reached at