A South-American Head Figure 4. Confocal microscopic appearance of ancient scalp hair: (a) Extracellular polymeric substances (EPS) on hair surface of TSA nu1 (arrows); (b) EPS on hair surface of a naturally mummified specimen. The confocal microscopic features of these bacterial products are indistinguishable in the two specimens. doi:10.1371/journal.pone.0002053.g004 result in the accumulation of significant amounts of toxic elements and thus may, occasionally, mislead criminal investigators. Materials and Methods Biopsies were taken from the scalp, the submandibular area and at the wound’s edge. Skin and muscle samples were used for immunohistochemical analyses and examined by light and electron microscopy. Immunohistochemistry was used to check the preservation of tissue antigenicity. Samples were fixed in neutral buffered formalin, embedded in paraffin, and processed by hand. Biopsies were examined in haematoxylin-eosin (H& E) stained sections. In addition to H & E, other histochemical methods such as periodic-acid Shiff reaction (PAS), Grocott (silver methenamine), elastic fiber stain and MSB trichrome were also used. Immunoperoxidase stains were performed on de-waxed paraffin sections using an avidin-biotin horseradish peroxidase technique. Antibodies identifying epithelia, nerves, muscles and blood vessels were applied to the tissue sections after suitable antigen retrieval methods [16,17] (Table 1 & Fig. 2). Hair and skin samples taken from the occipital area were used for Atomic Mass Spectrometry radiocarbon dating [18,19,20]. Samples of the ‘waxy’ material filling the skull were analysed using a LEO 1430 VP scanning electron microscope (LEO Figure 5. Transmission electron microscopy of ancient scalp hair: (a) EPS on TSA nu1 hair. Note bacterial and fungal profiles; (b) EPS surrounding a bacterium on the surface of another mummy’s hair, supporting the bacterial origin of the amorphous material. doi:10.1371/journal.pone.0002053.g005 Electron Microscopy Ldt, Cambridge, UK) with a Link ISIS 300 dispersive X-ray analyzer (EDX) equipped with the CameoTM program for X-ray colour imaging (Oxford Instruments, High Wycombe, UK) [21], directly in variable pressure. Table 2. ICP/MS elemental analysis of hair taken from TSA nu1, a naturally mummified South-American specimen and a contemporaneous sample. Elements TSA nu1 Naturally mummified Contemporaneous hair Arsenic Calcium Mercury Potassium Sodium Lead Zinc Magnesium 520 38 3.700 6410 780 1600 8680 1600 3 8.900 ,1 14.500 2.300 5.760 200 360 0.03–3 0.4–1.2 5–29 130–220 Note the enormous levels of lead, mercury, zinc and arsenic in our study specimen compared to the naturally mummified and contemporaneous specimens. Calcium, potassium, magnesium and sodium are, by contrast, much increased in the hair of the naturally mummified specimen. Values are expressed in mcg/g of hair; = not applicable. doi:10.1371/journal.pone.0002053.t002 PLoS ONE | www.plosone.org 5 April 2008 | Volume 3 | Issue 4 | e2053