Scanning Electron Microscopy in Forensic Science Background  Scanning electron microscopes (SEM) produce high magnification, low resolution images. Unlike traditional optical microscopes that use light and objective lenses to magnify a sample, SEM uses a focused, high-energy beam of electrons and an electromagnetic condenser to form an image. SEMs are capable of exceeding magnifications of 100,000X and resolutions below 1nm (Goldstein et al., 2003). Electrons are generated in the electron gun. Electrons leaving the gun, or cathode, are pulled down towards the anode and sample by a strong electric field. After passing though the anode, the electrons are condensed into a fine beam where they reach and pass through the objective lens to meet the sample (JoVE, 2018). The interaction between the incident electrons and the sample produce three primary signals: secondary electrons (SE), backscattered electrons (BSE), and X-rays (Vernon-Parry, 2000). The three signals carry different sample information. The individual signals are recognized by their respective detectors and, depending on what information is desired, are processed into an image. SEs, BSEs, and X-rays originate from disparate sample regions; therefore, they provide different sample information. SEs emanate from atoms on the sample’s surface, and produce detailed, topographic micrographs with high-spatial resolution. SE micrographs elucidate the overall morphology and unique, microscopic characteristics of a sample. A three-dimensional image can be produced when two micrographs, taken from different angles, are viewed through a stereoviewer (Taylor, 1973) or through specialty software (Huges et al., 2005). Unlike SEs, BSEs do not originate from the sample. BSEs are refracted incident electrons. Some incident electrons are able to pierce just below the sample’s surface where they collide with atoms within the sample.  Atoms within the sample are much larger than the incident electrons, and consequently, the incident electrons are “pushed” or “bounced” out of the sample. The more massive an element is, that is to say, the larger the atomic number, the more the incident electrons are scattered upon impact. Visually, more massive atoms appear brighter than less massive atoms. Thus, when compared to a known sample, an unknown sample’s composition can be tentatively determined.Characteristic X-rays are formed when incident electrons remove an inner-shell electron from an atom in the sample. True to the Aufbau principle, electron(s) from the outer-most shell replace the displaced inner-shell electron(s). In doing so, the outer-most electron must release energy. It does so in the form of X-rays. The energies, and consequently wavelengths, of the X-rays are directly related to the element(s) present, and can be used to perform compositional analysis on the sample. X-rays are detected by an energy-dispersive spectrometer (EDS or EDX) or by a wavelength-dispersive spectrometer (WDS).Sample preparation varies depending on the sample material, desired information, and type of SEM used. Most biological samples typically have a higher concentration of water compared to non-biological samples. “Wet” samples must be dried prior to viewing, as the SEM vacuum can damage the sample during evacuation by improperly dehydrating the sample. Moreover, this leeching of water can contaminate the instrument. Drying of the sample should be done in such as way as to preserve the sample’s natural integrity. Samples must also be conductive, either inherently or by means of conductive coating. SEM is a powerful, interdisciplinary tool. Forensic scientists are attracted to this instrument due to its high magnification and low resolution capabilities, as well as its ability to perform elemental analysis. Forensic scientists use SEMs to detect forgeries, determine the cause of textile damage, and detect the presence of GSR (Krüsemann, 2001).History of SEM in Detecting GSRPulling the trigger of a firearm causes the firing pin to hit and activate the primer, which is located on the bottom of the cartridge. This, subsequently, ignites the gunpowder and propels the bullet out of the gun’s barrel. The combustion of the gunpowder results in the production of immense heat and pressure, vaporizing metals from the primer and cartridge. Immediately out of the gun, the particulates begin to cool and condense. These particulates are commonly referred to as gunshot residue (GSR), but they may also be referred to as firearm discharge residue (FDR) or cartridge discharge residue (CDR). Cartridge primers, and therefore GSR, consist primarily of three compounds: lead styphnate (Pb), barium nitrate (Ba), and antimony sulfide (Sb). Finding GSR on an individual is not enough to find the suspect guilty of firing a weapon. Studies have found GSR as far as 13.5 m away from the shooter (Gerard et al., 2011). Additionally, studies have shown objects and individuals close to the bullet’s trajectory are exposed to similar amounts of GSR as the shooter (Gerard et al., 2011). However, forensic scientists have shown that GSR is not found unless a weapon has been fired (McCullough & Niewoehner, 2009).In 1933, Teodoro Gonzales developed the dermal nitrate test (Cowan & Purden, 1976). This test was able to identify the presence of barium. Briefly, a suspect’s hand was coated with melted paraffin. Once cooled, the hardened paraffin was removed, taking with it any GSR present. The mold was sprayed with a solution of N,N’-diphenyl-benzidine and sulfuric acid. A blue coloration indicated a positive result. The dermal nitrate test was not specific for barium nitrate in GSR and would yield a positive result if nitrates from organic substances were present (Romolo & Margot, 2001). A more comprehensive, reliable test for GSR was introduced in 1959 (Harrison & Gilroy, 1959). This procedure was able to detect barium and antimony but not lead. Additionally, this test was relatively insensitive (Romolo & Margot, 2001). Neutron activation analysis (NAA) was used in 1964 to identify barium and antimony in GSR (Ruch et al., 1964). NAA was sensitive and reliable, but was unable to detect the presence of lead. Additionally, NAA required a highly trained individual to run the nuclear reactor (Romolo & Margot, 2001).In 1968, whispers of GSR detection by SEM/EDX were coming from England (Heard, 2011); however, it would not be until 1976 that the first literature about using SEM for detection of GSR would appear (Nesbitt, Wessel, & Jones, 1976). The next 20 years saw the use of varying techniques for the detection of GSR (Romolo & Margot, 2001) such as: atomic absorption spectroscopy (Krishnan, 1971), proton-induced X-ray emission (Sen et al., 1982), inductively coupled plasma atomic emission spectroscopy (Koons, Havekost, & Peters, 1998), and plasma-mass spectrometry (Koons, 1998). Only one of these techniques allowed for the specific identification of lead, barium, and antimony from GSR: SEM/EDX.SEM/EDX and GSRSEM/EDX is a well-accepted method for detecting and verifying the presence of GSR and provides morphological data and the elemental composition of samples (ASTM Standard, 2010). Micrographs are generated using BSEs to identify potential GSR particulates, Pb, Ba, and Sb. Most SEM/EDX instruments used for detecting GSR are automated to some degree. Once identified, an SEM program stores the GSR candidate’s location on the sample stub. After the instrument has analyzed the entire stub, a trained analyst reviews the potential GSR particulates. Samples that are further suspected of being GSR particulates undergo EDX analysis to determine the particulate’s elemental composition. GSR particulates have a spherical morphology and appear burnt or melted (Figure 1). EDX analysis of particulates can show a sample’s elemental composition (Figure 2).       Figure 1: BSE micrograph of GSR particulate. Image taken without permission from Romolo. & Margot, Identification of gunshot residue: a critical review (2001).        Figure 2: EDX analysis of GSR particulate. The analysis show the particulate is made up of lead, antimony, and barium atoms. Image taken without permission from Romolo. & Margot, Identification of gunshot residue: a critical review (2001).  The combination of BSE micrographs and EDX analysis provide specific, accurate methodologies for forensic scientists to confidently decide if a particulate is or is not gunshot residue. While these techniques cannot differentiate shooter from bystander, SEM/EDX can determine if a suspect was near a firearm when it was discharged.Literature CitedASTM Standard. E1588-10e1. (2010). Standard guide for gunshot residue analysis by scanning electron microscopy/energy dispersive X-ray spectrometry. ASTM International, West Conshohocken, PA. https://compass.astm.org/download/E1588-10e1.12595.pdfCowan, M., & Purdon, P. (1967). A study of the paraffin test. Journal of Forensic Sciences, 12(1), 19.Gerard, R., McVicar, M., Lindsay, E., Randall, E., & Harvey, E. (2011). The long range deposition of gunshot residue and the mechanism of its transportation. Canadian Society of Forensic Science Journal, 44(3), 97-104.Goldstein, J., Newbury, D., Joy, D., Lymon, C., Echlin, P., Lifshin, E., Sawyer, L., Michael, J. (2003). Scanning Electron Microscopy and X-ray Microanalysis. 3rd Ed. Springer, New York, NY.Harrison, H., & Gilroy, R. (1959). Firearms discharge residues. Journal of Forensic Science, 4(2), 184-199.Heard, B. (2011). Handbook of firearms and ballistics: examining and interpreting forensic evidence (Vol. 1). John Wiley & Sons.Hughes, L., Archer, C., & Ap Gwynn, I. (2005). The ultrastructure of mouse articular cartilage: collagen orientation and implications for tissue functionality. A polarised light and scanning electron microscope study and review. European Cells & Materials, 9(68), e84.JoVE Science Education Database. Analytical Chemistry. Scanning Electron Microscopy (SEM). (2018). JoVE. Cambridge, MA. https://www.jove.com/scienceeducation/5656/scanning-electron-microscopy-semKoons, R. (1998). Analysis of gunshot primer residue collection swabs by inductively coupled plasma-mass spectrometry. Journal of Forensic Science, 43(4), 748-754.Koons, R., Havekost, D., & Peters, C., (1988). Determination of barium in gunshot residue collection swabs using inductively coupled plasma-atomic emission spectrometry. Journal of Forensic Science, 33(1), 35-41.Krishnan, S., (1971). Rapid detection of firearms discharge residues by atomic absorption and neutron activation analysis. Journal of Forensic Science. 16. 144-151.Krüsemann, H. (2001). SEMs and forensic science. Problems of Forensic Sciences, 47, 110-121.McCullough, J., & Niewoehner, L. (2009). A European study of the prevalence of GSR in random population and selected professional groups. lecture, SCANNING.Nesbitt, R., Wessel, J., & Jones, P. (1976). Detection of gunshot residue by use of the scanning electron microscope. Journal of Forensic Science, 21(3), 595-610.Romolo, F., & Margot, P. (2001). Identification of gunshot residue: a critical review. Forensic Science International, 119(2), 195-211.Ruch, R., Buchanan, J., Guinn, V., Bellanca, S., & Pinker, R. (1964). Neutron Activation Analysis in Scientific Crime Detection–Some Recent Developments. Journal of Forensic Sciences, 9.Sen, P., Panigrahi, N., Rao, M., Varier, K,  Sen, S., & Mehta, G. K. (1982). Application of proton-induced X-ray emission technique to gunshot residue analyses. Journal of Forensic Science, 27(2), 330-339.Taylor, M. (1973). Scanning electron microscopy in forensic science. Journal of the Forensic Science Society, 13(4), 269-280.Vernon-Parry, K. (2000). Scanning electron microscopy: An introduction. III-Vs. Review, 13(4), 40-44.