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.     

Practical Considerations in Trace Element Analysis of Bone by Portable X‐ray Fluorescence

Forensic anthropologists are more often turning to nondestructive methods to assist with skeletal analyses, specifically for trace elemental analyses. Portable XRF (pXRF) instruments are versatile and are able to be used in diverse settings or for specimens of a shape and size that cannot be accommodated by laboratory‐based instruments. Use of XRF requires knowledge of analysis parameters such as X‐ray penetration and exit depth. Analysis depth was determined by examining pure elements through known thicknesses of equine bone slices. Correlation between the element's X‐ray emission energy and the depth of reading was observed. Bone surfaces from a small unknown historic cemetery were analyzed before and after sanding of the periosteal surface to observe possible changes in XRF readings based on potential diagenesis. Results validate the pXRF device as a powerful and convenient instrument for nondestructive analysis, while highlighting limitations and considerations for the analysis of osseous materials.     

Determining Volumetric Shrinkage Trends of Burnt Bone Using Micro-CT

Understanding the degree and pattern of shrinkage undergone by bone when subjected to heating is crucial to accurately deduce a biological profile from incinerated remains. X-ray microtomography (micro-CT) enables a nondestructive insight into hard tissue structural changes, while allowing for an accurate documentation of volumetric and trabecular shrinkage. Sheep ribs were experimentally burned at temperatures between 400 and 1000°C in 100°C increments and their volumetric shrinkage was calculated. Observed shrinkage ranged from 14.0% at 400°C to 45.5% at 1000°C. Bones burned at temperatures up to 600°C showed no significant difference, whereas the 700 and 800°C samples exhibited higher shrinkage. Bones burnt at 900 and 1000°C showed significantly higher shrinkage than the other temperature groups. Findings signify the potential of micro-CT in research on the effects of factors such as diagenesis or burning on the bone density, morphology and microarchitecture.