Nine milligrams of dental calculus was selected for sonication in ultrapure water. Before sonication, the sample was first washed in ultrapure water to remove loose soil and other surface debris. The calculus was then further decontaminated as follows. First, the sample of calculus was placed under a stereomicroscope at a magnification of up to 50× and inspected for contaminants. A fine sterile acupuncture needle was used in conjunction with a 0.6 M solution of HCl to clean the external facets of the calculus. Once completely free of any visible surface contaminants, the calculus was washed again in ultrapure water and then placed in a sterile microcentrifuge tube containing ultrapure water to remove any residual HCl. The sample was then subjected to controlled sonication for approximately 6 min until the calculus was reduced into a fine powder. The calculus powder suspension was transferred to two microscope slides and evaporated under controlled conditions. These steps were performed in a laboratory at the University of York dedicated to the extraction and mounting of microfossils from ancient dental calculus; no other material type or reference pigments were prepared in this laboratory.

Optical microscopy was initially performed at the Laboratory of Microarchaeology at the University of York, where the particles were counted and measured under transmitted and polarized light using a Zeiss Axio Scope.A1 binocular compound microscope equipped with motorized stage. Mean particle size and SD were determined by measuring 100 blue particles using AxioVision image software (data file S1). Slides were then analyzed at the microscope facilities of the Department of Archaeology at the Max Planck Institute for the Science of Human History (MPI-SHH) using an Olympus BX53M equipped with transmitted light, cross-polarized light, differential interference contrast filters, and an Olympus 20.7 Mpx camera. After locating and photographing the blue particles, a high-resolution map was made for one of the slides to aid in the targeting blue particles for SEM-EDS analysis (fig. S5F).

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