Scanning electron microscopy analysis of experimental bone hacking trauma

The authors report on their macro- and microscopy study of bone lesions made by a sharp force instrument (a single blade knife), and a sharp-blunt instrument classified as a chopping weapon (a hatchet). The aim of this work was to attempt to identify the instrument by analyzing the general class characteristics of the cuts. Each weapon was used on human bones. The results indicate that macroscopic analysis is more problematic. The microscopic analysis assessed that characteristics examined were effective in distinguishing sharp from sharp-blunt injury to the bone. The microscope facilitates analysis unachievable with macroscopic methods, some three-dimensional characteristics not visible to the naked eye being clearly defined with its use. Emphasis has been placed on the value of SEM as an anthropologist's tool in bone lesion injuries.     

Scanning Electron Microscope Analysis of Gunshot Defects to Bone: An Underutilized Source of Information on Ballistic Trauma

Recent years have seen increasing involvement by forensic anthropologists in the interpretation of skeletal trauma. With regard to ballistic injuries, there is now a large literature detailing gross features of such trauma; however, less attention has been given to microscopic characteristics. This article presents analysis of experimentally induced gunshot trauma in animal bone (Bos taurus scapulae) using full metal jacket (FMJ), soft point (SP), and captive bolt projectiles. The results were examined using scanning electron microscopy (SEM). Additional analysis was conducted on a purported parietal gunshot lesion in a human cranial specimen. A range of features was observed in these samples suggesting that fibrolamellar bone response to projectile impact is analogous to that observed in synthetic composite laminates. The results indicate that direction of bullet travel can be discerned microscopically even when it is ambiguous on gross examination. It was also possible to distinguish SP from FMJ lesions. SEM analysis is therefore recommended as a previously underexploited tool in the analysis of ballistic trauma.     

Quantitative analysis of sharp-force trauma: an application of scanning electron microscopy in forensic anthropology.

Scanning electron microscopy (SEM) has occasionally been used by anthropologists and forensic scientists to look at morphological characteristics that certain implements leave on bone. However, few studies have addressed techniques or protocols for assessing quantitative differences between tool marks on bone made by different bladed implements. In this study, the statistical variation in cut mark width was examined between control and test samples on bone using a scalpel blade, paring knife, and kitchen utility knife. Statistically significant differences (p < .0005) were found between cut marks made by the same knife under control and test conditions for all three knife types used in the study. When the control sample and test samples were examined individually for differences in mean variation between knife types, significant differences were also found (p < .0005). While significant differences in cut mark width were found, caution should be used in trying to classify individual cut marks as being inflicted by a particular implement, due to the overlap in cut mark width that exists between different knife types. When combined, both quantitative and qualitative analyses of cut marks should prove to be more useful in trying to identify a suspect weapon. Furthermore, the application of SEM can be particularly useful for assessing many of these features.     

SEM and Stereomicroscopic Analysis of Cut Marks in Fresh and Burned Bone

This study documents the prevalence of cut mark characteristics in fresh and burned domestic pig ribs. Stab wounds from single edge serrated and smooth‐edged knives were inflicted in the vertebral and sternal regions of each fresh rib. Each rack of ribs was then divided into vertebral and sternal units. Vertebral units were defleshed and their associated cut marks were examined using a stereomicroscope. Sternal units were burned in an outdoor fire pit and examined with the addition of a scanning electron microscope (SEM). Linear cuts, V‐shaped cross‐sections, mounding, hinge fractures, and wastage were all observed on burned ribs. There was an overall decrease in the prevalence of all features (up to a 40% decrease), regardless of knife type, in burned ribs. Striations within cut marks were not observed in either fresh or burned ribs. Oblique faulting and bone lifts could only be observed using the SEM. Mounding and wastage were obliterated during the burning process. Therefore, cut marks in burned bone should ideally be examined for their characteristics utilizing an SEM.     

Assessment of Wear‐Related Features of the Kerf Wall from Saw Marks in Bone*†

Abstract: Analyses of saw marks in bone may yield important information about the class characteristics of saws used in postmortem dismemberments, yet little research has been directed at identifying saws’ individualizing characteristics. This study adds to existing saw mark analysis methodologies by examining wear‐related features of kerf walls using light‐ and environmental scanning electron microscopy. A crosscut saw and hacksaw were used to create sequences of 30 cuts in bone; these sequences reveal patterns of progressive loss of fine details of kerf wall morphology with increasing saw blade wear, because of the rounding of sharp points and edges. Nevertheless, diagnostic kerf wall features used to establish class characteristics persist despite these wear‐related changes. Unsuccessful attempts at statistical analysis of wear‐related changes, based on striae width and density, suggest these patterns are not readily quantifiable. Additionally, despite the scanning electron microscope’s superior imaging capabilities, it provided few practical, methodological gains over traditional light microscopy.     

Gunshot residues on dry bone after decomposition-a pilot study

Abstract: Very little literature exists concerning radiochemical and microscopic analyses of gunshot wounds in decomposed material, and even less concerning skeletonized samples; the most advanced technologies may provide useful indications for the diagnosis of suspect lesions, especially if gunshot wounds are no longer recognizable. However, we know very little of the survival of gunshot residues (GSR) in skeletonized samples. This study examined nine gunshot wounds produced on pig heads which then underwent skeletonization for 4 years, and four gunshot entries on human heads from judicial cases which were then macerated to the bone in water; the samples underwent scanning electron microscopy coupled with energy dispersive X‐ray (SEM‐EDX) analysis. Positive results for GSR were observed only in four of the nine animal samples and in all four human samples. Among the human samples, two lesions showed Pb and Sb, one lesion only Pb, and one Pb, Sb, and Ba. This pilot study showed the survival of GSR in skeletal material and therefore the crucial importance of SEM‐EDX analyses on skeletonized material. Further studies are needed in order to ascertain the role of environmental modifications of GSR.     

Macroscopic and microscopic analysis of knife stab wounds on fleshed and clothed ribs

Stab wounds upon bone are analyzed to interpret the weapon used and the physical context in which the attack occurred. The literature demonstrates that most research conducted pertaining to wound patterns has been carried out on defleshed and unclothed bone samples, not adequately replicating actual circumstances. For this research, six half pig torsos (Sus scrofa), fleshed (including muscle, fat, epidermis, and dermis layers) and clothed, were stabbed using three knife types, applying both straight and downward thrusts. Analysis conducted macroscopically and through a scanning electron microscope with an environmental secondary electron detector revealed a general lack of consistency in wound pattern and associated secondary effects. Consequently, it was not possible to establish wound pattern per knife type as suggested in previous research or relate it to stab motion. Advantage of microscopic analysis was evident in recognizing wound traits and observation of trace evidence not visible macroscopically.     

Interpreting the Effects of Burning on Pre-incineration Saw Marks in Bone

This study examined the effects of fire on the features associated with saw marks in bone. Both class and individual characteristics were examined using stereomicroscopy and scanning electron microscopy (SEM). Twenty-four semifleshed Sus scrofa L. tibiae were sawed into three sections with the middle section having deep and shallow false starts. Twelve saw blades of varying age and type were each used to cut two tibiae. In each case, the first tibia was burned in an outdoor open fire to the point of partial calcination. The second tibia, our control, was macerated using a heated enzyme solution. Controls and burned specimens were examined for the following characteristics: breakaway spur, tooth hop, false start, exit chipping, tooth imprint, breakaway notch, pull out striae, kerf flare, and blade drift. In general, there was parity in the observed characteristics in the burned samples using the SEM and the stereomicroscope. SEM observation, however, provided enhanced images, with the addition of observing individual tooth imprints, previously not visible. Therefore, this study recommends using an SEM for the examination of saw cuts in burnt bone.     

Unusual injuries caused by blows with a sash saw

A 62-year old man was found killed by multiple blows against the face and head; near the victim a hammer with broken grip, a bended saw (weight 142 g), and in the possession of the doer two hatchets. Some straigt, parallel, horizontal wounds in the face with smooth lips and straigt incisions of the maxilla first were interpreted as violations by hatchet. The culprit confessed attacks by hammer and saw, but not by hatchet. Experimental blows with a saw like the used on the leg of a corpse showed an unexpected result: it was possible to produce wounds of the soft-tissues and the bone similar to those by hatchets.     

A method for studying knife tool marks on bone

The characteristics of knife tool marks retained on hard tissues can be used to outline the shape and angle of a knife. The purpose of this study was to describe such marks on bone tissues that had been chopped with knives. A chopping stage with a gravity accelerator and a fixed bone platform was designed to reconstruct the chopping action. A digital microscope was also used to measure the knife angle (θ) and retained V-shape tool mark angle (ψ) in a pig skull. The κ value (elasticity coefficient; θ/ψ) was derived and recorded after the knife angle (θ) and the accompanied velocity were compared with the proportional impulsive force of the knife and ψ on the bone. The constant impulsive force revealed a correlation between the V-shape tool mark angle (ψ) and the elasticity coefficient (κ). These results describe the tool marks--crucial in the medicolegal investigation--of a knife on hard tissues.     

Morphological observation of puparia of Chrysomya nigripes (Diptera: Calliphoridae) from human corpse

This article presents Chrysomya nigripes Aubertin as a blow fly species of forensic importance in Thailand, and morphological observation of fly puparia using scanning electron microscopy (SEM). Morphologically, we focused on the characteristics of puparia used to accurately identify fly species. Numerous puparia of C. nigripes were found aggregated, adhering side by side, on the tibia of a skeletonized corpse, which was recovered from a forested area of Chiang Mai, northern Thailand. In the triangular shape of the anterior end of the puparia, three thoracic segments and broad hairy patches beginning dorsolaterally at the sixth segments were distinguishing characteristics. This study, showing pupariation of the flies along the bone of a corpse as well as morphological features, provides important guidance in identifying C. nigripes puparia. A key to differentiate puparia of C. nigripes from the other flies of forensic importance in Thailand is given.     

Validity of the use of porcine bone in forensic cut mark studies

Porcine bone is often used as a substitute for human bone in forensic trauma studies, but little has been published on its comparative mechanical behavior. The factors affecting mechanical properties and therefore selection of bone models are complex and include the age of the animal at death, and physiological loading conditions, the latter being of particular relevance when using a quadrupedal animal as a human substitute. The regional variation in hardness of adult and infant porcine bones was investigated using Vickers’ indentation tests and compared to published data for human limb bones to relate differences to inherent genetic effects and loading influences, and to examine the validity of the porcine‐human model. Significant differences in hardness were observed both along and around the adult porcine humerus and femur, but no significant differences were found along the length of the infant bones. Significant differences were found between the forelimb and hindlimb, but only in the infant specimens. The hardness values for porcine adult cortical bone from the femur (52.23 ± 1.00 kg mm^?2) were comparable to those reported in the literature for adult human cortical bone from the fibula, ilium, and calcaneus. These data will help inform subject selection in terms of both species and bone type for use in future trauma studies.     

Determining Postmortem Interval Using Glycoproteinous Adhesion Deposits by Balanus improvisus on Human Skeletal and Dental Remains

An anthropological analysis was conducted on skeletal and dental remains brought to the Galveston County Medical Examiner's office. The skeletal remains were dry, fragmented, and absent of typical fluvial characteristics. During microscopic examination, semitransparent, circular objects were discovered on the dentition, the mandible, tibial plateau, and distal femur. The objects were glycoproteinous adhesions deposited by the acorn barnacle, Balanus improvisus. B. improvisus is an intertidal barnacle found in estuaries in Galveston Bay. Basal diameter of the adhesions on the dentition were significantly smaller than those found on the postcranial bones (p = 0.010), indicating two consecutive cohorts adhered to the bone and dentition. As settlement typically occurs once a year, this would indicate that the remains were in the fluvial environment for at least 375–410 days. It is important in geographic areas that have prevalent fluvial environments that human remains, particularly dentition, are microscopically examined for marine life evidence.     

Microscopic Indicators of Axe and Hatchet Trauma in Fleshed and Defleshed Mammalian Long Bones*

Abstract: The characterization of wounds in bone caused by chopping weapons has been based on either semi‐fleshed or defleshed specimens. This approach has not been adequately justified as reflecting actual cases involving fleshed bone. Likewise, the histological appearance of features in chopping wounds also deserves further attention. We used 11 fresh pig (Sus scrofa) articulated hind limbs, including the femur, tibia, and fibula with contiguous surrounding flesh (including an intact epidermal layer), to receive wounds using two axes and two hatchets. Scanning electron microscopy analysis of these wounds exhibited osteon pullouts in the fracture surfaces of fleshed specimens, suggesting the attenuation of force by the surrounding flesh. Lamellar separation was also exhibited at the impact sites and fracture surfaces of both fleshed and defleshed specimens. A consistently rough morphology is characteristic of fracture surfaces while impact surfaces are smooth and yielded evidence of striations from each implement.     

Differentiation of traumatic and heat-induced dental tissue fractures via SEM analysis

Previous studies have examined the effects of heating on teeth; however, none have identified characteristics that allow analysts to differentiate traumatic from heat-induced fractures. This study examined our ability to discern notable differences in preincineration traumatic fractures and heat-induced fractures in postincineration dentition. Twelve anterior dental specimens were subjected to blunt force trauma while a second set were not. All 24 samples were then incinerated in a muffle furnace at a peak temperature (900°C) consistent with house fires. The specimens were subsequently examined with a scanning electron microscope to identify and compare heat-induced and traumatic fractures. The results obtained during examination yielded no differences between the features displayed by specimens that had been inflicted with preincineration trauma and those that did not. Unlike bone, distinguishing features for the differentiation of traumatic and heat-induced fractures could not be compiled.     

Long Bone (Humerus,Femur, Tibia) Measuring Procedure in Cadavers

In this work, we present a measuring methodology for long bones of the limbs (humerus, femur, and tibia) of human corpses. Measurements of cadaveric height and long bone lengths were conducted on 72 corpses (20 females and 52 males) from the School of Medicine at the National Autonomous University of Mexico. Additionally, these measurements were compared with those taken from dry bones of a subsample of individuals. Our results show marginal differences (TEM% = 0.59) between cadaveric and dry bone measurements, resulting from different osteometric technical procedures. This note outlines the measuring methodology, which will be subsequently used to create regression formulas for stature estimation.     

Frozen human bone: a microscopic investigation

The taphonomic effects of prolonged extreme cold and freezing on human bone have received little research attention. Questions of specific interest include whether previously frozen bone can be identified and whether freezing alters the structural integrity enough to prevent histological aging. There is no evidence from previous studies that freezing damages the structural integrity, and to date no research investigating the freezing process on bone microstructure has been undertaken. This research attempts to distinguish histologically previously frozen bone from nonfrozen bone by identifying patterned defects. To determine the effects of freezing in bone microstructure using light and scanning electron microscopy (SEM), several human bone sections were subjected to prolonged freezing and allowed to thaw before thin sectioning. Light microscopy failed to demonstrate statistically significant differences between frozen and nonfrozen specimens. SEM analysis revealed fractures, although these lacked pattern and did not occur systematically throughout the section. Evidence of microstructural changes caused by liquid expansion, however, was remarkable but did not alter the structural integrity of the microstructure. The results of this study suggest that freezing does not alter the process of histomorphological analysis.     

Chemical Differentiation of Osseous and Nonosseous Materials Using Scanning Electron Microscopy–Energy-Dispersive X-Ray Spectrometry and Multivariate Statistical Analysis

Identification of osseous materials is generally established on gross anatomical features. However, highly fragmented or taphonomically altered materials may be problematic and may require chemical analysis. This research was designed to assess the use of scanning electron microscopy–energy-dispersive X-ray spectrometry (SEM/EDX), elemental analysis, and multivariate statistical analysis (principal component analysis) for discrimination of osseous and nonosseous materials of similar chemical composition. Sixty samples consisting of osseous (human and nonhuman bone and dental) and non-osseous samples were assessed. After outliers were removed a high overall correct classification of 97.97% was achieved, with 99.86% correct classification for osseous materials. In addition, a blind study was conducted using 20 samples to assess the applicability for using this method to classify unknown materials. All of the blind study samples were correctly classified resulting in 100% correct classification, further demonstrating the efficiency of SEM/EDX and statistical analysis for differentiation of osseous and nonosseous materials.     

Use of laser-scanning confocal microscopy in the detection of diagenesis in bone

This research demonstrates the value of laser scanning confocal microscopy (LSCM) as a research tool in osteological studies, and diagenetic studies in particular. LSCM combines properties of light and scanning electron microscopy using laser light to excite fluorophores throughout the z-axis, developing a 3-D image. Using differential staining and selecting for specific wavelengths of light, one can image targeted materials. This research is divided into two parts: visualizing bone structures such as proteins and their decompositional products and visualizing diagenesis. Part one of this study utilized pig bones as a means of testing the overall ability of LSCM to fluoresce bone. Twenty-three samples were imaged, including 13 samples from a decompositional study conducted 5 years previous, and 10 “fresh” samples collected from a commercial butcher. This part of the study determined that protein and organic components of the bone could be fluoresced and diagenetic alteration could be imaged. The second part of the study used human samples as a means of imaging and mapping diagenetic alterations. The second part of the study used 13 samples, including 4 clinical, 7 ancient, and 2 modern controls. The pig study used Basic Fuchsin and SlowFade Gold stains, while the human study used toluidine blue. Images were also taken with unstained elements. The results of the non-human study found that a fresh bone fluoresced differently than that of a 5-year subset, while the results of the human study confirmed these findings and determined that the bone diagenesis can be mapped using LSCM.     

Analysis of microtraces in invasive traumas using SEM/EDS

Scanning electron microscopy in combination with energy-dispersive X-ray spectrometry (SEM/EDS) is a proven forensic tool and has been used to analyze several kinds of trace evidence. A forensic application of SEM/EDS is the examination of morphological characteristics of tool marks that tools and instruments leave on bone. The microtraces that are left behind by these tools and instruments on the bone are, however, often ignored or not noticed at all. In this paper we will describe the use of SEM/EDS for the analysis of microtraces in invasive sharp-force, blunt-force and bone-hacking traumas in bone. This research is part of a larger multi-disciplinary approach in which pathologists, forensic anthropologists, toolmark and microtrace experts work together to link observed injuries to a suspected weapon or, in case of an unknown weapon, to indicate a group of objects that could have been used as a weapon. Although there are a few difficulties one have to consider, the method itself is rather simple and straightforward to apply. A sample of dry and clean bone is placed into the SEM sample chamber and brightness and contrast are set such that bone appears grey, metal appears white and organic material appears black. The sample is then searched manually to find relevant features. Once features are found their elemental composition is measured by an energy dispersive X-ray spectrometer (EDS). This method is illustrated using several cases. It is shown that SEM/EDS analysis of microtraces in bone is a valuable tool to get clues about an unknown weapon and can associate a specific weapon with injuries on the basis of appearance and elemental composition. In particular the separate results from the various disciplines are complementary and may be combined to reach a conclusion with a stronger probative value. This is not only useful in the courtroom but above all in criminal investigations when one have to know for what weapon or object to look for.