Kerf Patterning on Animal Cremains: Preliminary Analysis of Microscopy Methods Christopher E. Barrett 1 , Nambi Gamet 1 1 Anthropology Department, Western Washington University, 516 High St., Bellingham, WA 98225 Abstract Introduction Materials & Methods Results Discussion In Forensic science reconstruction methodologies are critical to the assessment and authentication of human behavior after time of action. Archeological samples can present evidence of burning and thus provide time depth to the issues involved (Ubelaker, 2009). Fire has been a common method for the destruction of evidence in homicides, accidental deaths, bombings, and aircraft accidence (Porta et al., 2013; Ubelaker, 2009; Alunni et al., 2014). Fire can be employed to destroy forensic evidence in order to mislead or remove identification and reconstruction of behavior. Contemporary research and case studies have greatly augmented knowledge regarding the effects of extreme heat on incinerated remains or cremains. Resulting from these scholarly efforts, enhanced interpretation is now possible on such issues as: • the extent of recovery • reconstruction • trauma • individual identification • color variation • DNA recovery Sharp force trauma and cut mark analyses to date have been intermittent and superficially researched across a range of disciplines, despite its potential to significantly contribute to anthropological investigation (Herrman and Bennett, 1999; Tennick, 2012;). The use of fire is an attempts to obscure a body is commonly encountered, however, fire does not necessarily destroy evidence of trauma on bone (Robbins et al., 2015). Advanced microscopy techniques such as scanning electron microscopy (SEM) may also provided enhanced observational power forensic reconstructions (Bartelink and Wiersema, 2001; Kooi and Fairgrieve, 2013; Marciniak, 2009; Robbins et al. 2015). Cremains are found within many broad anthropological contexts induced by both human behavior as well as potentially stochastic environmental events, adding to the challenge of reconstruction efforts (Alunni et al., 2013; Porta et al,. 2013) . Ostensibly, enhanced observational methodologies from developing x-ray and microscopy technologies, like SEM, have potential to remove limitations met by other forms of observation and reconstruction techniques, standard in forensics and anatomical methods. This study recommends using an SEM for the examination of saw cuts in burnt bone (Robbins, 2015). Archaeological field methods and research using broad remote sensing technologies demonstrate an emphasis on conservation as well as non- invasive non-destructive processes in sample extraction, preparation, and analysis. In culture resource management, archaeological excavation and surveying has political and corporate applications while relying on ecologically and sociocultural sensitive protocols. A social consciousness underrepresented in principle ecological, sociological, and behavioral research that is non-anthropological in origin. Enhanced observational techniques and methodologies are made possible with progressive equipment and technology. With additional observational information provided by advanced microscopy, there are increasing opportunities for multidisciplinary work. A frequently overlooked element in the analysis of burned human remains is reconstruction. Reconstruction provides a more holistic opportunity for morphological interpretation and can greatly facilitate determinations of human vs. non-human animal and recognition of specific skeletal elements. Reconstruction can also increase the probability of identification and recognition of trauma (Porta et al., 2013; Robbins et al., 2014; Rickman 2014; Ubelaker, 2002; Ubelaker, 2009). Limitations and future ideas: SEM images of unburnt samples were not taken, which would have provided further analyses for EDS spectrum comparisons prior to and after incinerating activity. Potential follow up studies may include EDS spectrum analyses of bones preserved in various preservation mediums. Reconstruction capabilities could be evaluated using metal residue analyses of metal blunt force trauma on bone. We investigate the utility of scanning electron microscope (SEM) methodologies in observing saw kerf patterning on burnt bone cut with different types of saws. SEM analysis of kerf walls provide observations that stereomicroscopes cannot. Kerf wall observations and interpretations on cremains found within archaeological and forensic contribute to SEM validity in methodologies of anthropological investigations. We divided one Bos taurus, one Equidae, and two Cervus elaphus long bones into three 9 cm segments using four different tools. Incineration of bone segments was completed using a fire pit. Temperatures were monitored using a Digi-Sense thermocouple thermometer. Thin sections were prepared from the cut portions of each segment after burning. Observations of kerf patterning were made using light and SEM. Fractures and kerf wall patterning were observed using two different microscopy methods. SEM provided further observations in comparison to stereomicroscopes of kerf wall characteristics in cremains. When comparing SEM and light microscopes the SEM provides a superior observational method for the observation of kerf patterning in cremains. With the SEM kerf pattern characteristics became very clear. Shallow false starts as well as individual striations are very clear when compared to the stereo-light microscope. The SEM also provided images of the heat induced fractures as well as fractures due to weathering otherwise not visible using standard light microscopy. Two SEM/EDAX analyses were taken, providing elemental compositions of the interior kerf floor and patterns as well as the superficial bone. Energy- dispersive X-ray spectroscopy (EDS) analyses differed between the two site. Kerf flooring, although observationally heterogeneous, yielded a homogenous EDS spectrum distribution. Figure 4. Reciprocating saw cutting by Bos taurus A. Fisheye image of burnt kerf mark. B. Photo of unburnt kerf mark using stereo-light microscope. C. Photo of burnt kerf mark using stereo-light microscope. D. Image of kerf mark and location of EDAX analysis. E. Image of kerf mark and EDAX analysis. F. EDS spectrum of kerf floor. G. EDS spectrum of superficial surface. A. B. C. D. E. F. G. Marisa Acosta, Peter Thut, Charles Wandler, Mike Etnier, and Sarah Campbell for constructive edits, sample collection, equipment acquisition, and technical laboratory support and training. Acknowledgments Figure 1. One drawback to the using scanning electron microscopy (SEM) is that it operates under vacuum and in many SEMs the samples must be rendered conductive to be viewed. This is often achieved by coating samples with a very thin layer of palladium and gold metal particles or carbon. However, there are a number of different types of SEMs which all have specific purposes, often associated with additional pieces of equipment like specialized stages or collectors. Some of these do not require dry or conductive samples. Fundamentally and functionally, electron microscopes are in many ways analogous to their optical counterparts (light microscopes: LM). This is somewhat surprising at first glance, given the contrast between the simple technology of the LM and the complex electronics, vacuum equipment, voltage supplies and electron optics system of electron microscopes. Figure 2. The formation of an image requires a scanning system to construct the image point-by-point and line-by-line. The scanning system uses two pairs of electromagnetic deflection coils (scan coils) that scan the beam along a line then displace the line position to the next scan so that a rectangular raster (represented here by a red circle instead) is generated both on the specimen and on the viewing screen. The first pair of scan coils bends the beam off the optical axis of the microscope and the second pair bends the beam back onto the axis at the pivot point of the scan. In order to produce contrast in the image the signal intensity from the beam-specimen interaction must be measured from point-to-point across the sample surface. Signals generated from the specimen are collected by an electron detector, converted to photons via a scintillator, amplified in a photomultiplier, and converted to electrical signals and used to modulate the intensity of the image on the viewing screen, seen in the different shades of grey on images D and E. Figure 3. After inner shell ionization, the atom may relax by emitting a Characteristic X-ray or an Auger electron. The energy of the Auger electron is related to the electronic configuration of the atom that was ionized by the primary electron beam, causing variation on the EDS spectrums seen in images F and G. The fluorescence yield is the relative yield or ratio of X-rays to Auger electrons, elements in the samples chemical composition and those hit with x-rays. Elements with low ionization energies, i.e. the lighter elements on the periodic table, have low fluorescence yields. That is, when an inner shell ionization occurs it is more likely that an Auger electron will be produced rather than an X- ray photon. This principle and shells are illustrated above. The intensities of X-ray peaks for elements of low atomic number are smaller compared to those with a higher fluorescence yield. Ammrf.org.au (2014). Introduction | MyScope. http://www.ammrf.org.au/myscope/confocal/introduction/ X-ray Absorption: Not all of the X-rays that are generated in the sample by the primary electron beam are emitted from the sample. This is particularly true in the SEM where X-rays are generated within the interactions at a depth of many microns. X-rays may be absorbed by other elements in the sample due to the photo-electric effect. This effect is the observation that many metals emit electrons when light shines upon them. Electrons emitted in this manner can be called photoelectrons. If the energy of an X-ray photon is equal to the critical ionization energy of an electron in another element in the sample then there is a high probability that the X-ray will be absorbed and a photoelectron produced. While the absorption of X-rays depends on the other elements present in the sample, it is also true that low-energy X-rays are more likely to be absorbed than those with higher energies, and elements with higher atomic numbers tend to be strong absorbers of lower energy X-rays. The length of the path that the X-ray travels through the sample will also influence absorption. The longer the path length, the more likely it is that the X-ray will be absorbed. Again, low-energy X-rays are more likely to be affected by longer path lengths than higher energy X-rays. Dividing one Bos taurus, one Equidae, and two Cervus elaphus bones were sawed once each with a circulating, reciprocating, and hand saw creating (n=16). Samples were prepared from sites of direct burning after five minutes of incineration with average temperatures of 476.2◦C recorded using a Digi-Sense thermocouple thermometer. Microscope observations and images of kerf patterning were completed and compared using light stereomicroscope and SEM. Energy dispersive X-ray analysis (EDAX) or energy dispersive X-ray microanalysis (EDXMA) is an analytical technique used for the elemental analysis and chemical characterization of samples, also quantifying levels of chemical residues. These analyses can vary depending on experimental and behavior manipulation like firing.