Assessing various Infrared (IR) microscopic imaging techniques for post-mortem interval evaluation of human skeletal remains

by Claudia Woess, Seraphin Hubert Unterberger, Clemens Roider, Monika Ritsch-Marte, Nadin Pemberger, Jan Cemper-Kiesslich, Petra Hatzer-Grubwieser, Walther Parson, Johannes Dominikus Pallua

Due to the influence of many environmental processes, a precise determination of the post-mortem interval (PMI) of skeletal remains is known to be very complicated. Although methods for the investigation of the PMI exist, there still remains much room for improvement. In this study the applicability of infrared (IR) microscopic imaging techniques such as reflection-, ATR- and Raman- microscopic imaging for the estimation of the PMI of human skeletal remains was tested. PMI specific features were identified and visualized by overlaying IR imaging data with morphological tissue structures obtained using light microscopy to differentiate between forensic and archaeological bone samples. ATR and reflection spectra revealed that a more prominent peak at 1042 cm^-1 (an indicator for bone mineralization) was observable in archeological bone material when compared with forensic samples. Moreover, in the case of the archaeological bone material, a reduction in the levels of phospholipids, proteins, nucleic acid sugars, complex carbohydrates as well as amorphous or fully hydrated sugars was detectable at (reciprocal wavelengths/energies) between 3000 cm^-1 to 2800 cm^-1. Raman spectra illustrated a similar picture with less ν₂PO₄³⁻at 450 cm^-1 and ν₄PO₄³⁻ from 590 cm^-1 to 584 cm^-1, amide III at 1272 cm^-1 and protein CH₂ deformation at 1446 cm^-1 in archeological bone material/samples/sources. A semi-quantitative determination of various distributions of biomolecules by chemi-maps of reflection- and ATR- methods revealed that there were less carbohydrates and complex carbohydrates as well as amorphous or fully hydrated sugars in archaeological samples compared with forensic bone samples. Raman- microscopic imaging data showed a reduction in B-type carbonate and protein α-helices after a PMI of 3 years. The calculated mineral content ratio and the organic to mineral ratio displayed that the mineral content ratio increases, while the organic to mineral ratio decreases with time. Cluster-analyses of data from Raman microscopic imaging reconstructed histo-anatomical features in comparison to the light microscopic image and finally, by application of principal component analyses (PCA), it was possible to see a clear distinction between forensic and archaeological bone samples. Hence, the spectral characterization of inorganic and organic compounds by the afore mentioned techniques, followed by analyses such as multivariate imaging analysis (MIAs) and principal component analyses (PCA), appear to be suitable for the post mortem interval (PMI) estimation of human skeletal remains.

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