GoKawiilSample
The nine species analysed in this study are listed in Supplementary Table 1. The 12 tooth samples, locations of origin, date, collection ID and precise tooth analysed are listed in Supplementary Table 2. Whenever possible, we selected lower first molars (LM1) and the mesio-buccal cusp, which experiences crushing and grinding forces and is the first to wear53,54 (Extended Data Fig. 2). When an LM1 was not available (for example, fossil samples), we used the functional cusp of the available molar or premolar, which is equivalent to an LM1’s mesio-buccal cusp in its masticatory function (Extended Data Fig. 2). In all cases, analysis was restricted to molar or premolar teeth and focused on the occlusal region proximal to the dentine horn to control for functional use and avoid worn enamel.
Histological sample preparation
Previous studies generated histological sections of PA, Arch1, Arch2, MH, Pt, Pp and Ca, but all used a standard set of histological methods55,56. Details of materials and consumables used for each sample can be found in their respective published articles (Supplementary Table 2). We identified no pathologies or taphonomic features in any sample. When wear was present, it was minimal and did not reach the dentine horn. All samples are permanent posterior teeth from adult individuals, with the exception of Ca, which was a subadult. Teeth were embedded in epoxy resin before being sectioned. Each tooth was sectioned through a plane intersecting the two mesial cusps (in the case of premolars, the two primary cusps; Extended Data Fig. 2) using a precision saw. The section was mounted on a microscope slide and used for other studies. For this study, we used the ‘off-cut’, which is the remaining portion of the tooth that was not mounted to the microscope slide. This off-cut was re-embedded, ground and polished as described below (‘Sample preparation for PEEM’).
The histological sectioning and analysis of the Eh sample was published in ref. 57; Vm was published in ref. 58; Hh, He and Pb were published in ref. 59. C. M. Dean led the histological preparation for these five samples, following standard protocols55,56. For these samples, no pathologies or taphonomic features were identified; when wear was present, it was minimal and did not reach the dentine horns.
Sample preparation for PEEM
Samples arrived sectioned; we re-embedded, then ground and polished them. For re-embedding, we soaked blocks or slides in anhydrous ethanol and placed them face-down in two-part plastic moulds (Ted Pella) and covered them with freshly mixed EpoFix (EMS; 25 g epoxy, 3 g hardener, mixed with a Thinky planetary mixer, mixing for 1 min and defoaming for 1 min. We cycled the moulds three times through vacuum with a diaphragm pump kit (Ted Pella) and vented with nitrogen, to remove ethanol, ensure full epoxy penetration and burst surface bubbles. EpoFix cured overnight in air.
All grinding and polishing used water as a coolant, but supersaturated with either 1 g l−1 CaCl 2 or 0.2 g l−1 Na 3 PO 4 (pH 9) in MilliQ water to prevent hydroxyapatite dissolution, dispensed by a peristaltic pump (Drive MFLX07522-30, Head MLFX07519-06, Tubing MFLX96410-14, all by MasterFlex, Avantor). We ground tooth surfaces with 600, 1,200 and 4,000 grit SiC paper (Buehler) using 5 N, 30 s each, 40/140 rpm co-rotating on a Buehler AutoMet 250 PM. We then polished the tooth surfaces on the same Buehler AutoMet 250 PM with 300-nm alumina (MicroPolish 0.3 µm, Buehler) using 10 N, 2 min, 40/140 rpm counter-rotating, then with 50-nm alumina (MasterPrep, Buehler) with 10 N, 1 min, 30/30 rpm counter-rotating. Both 300-nm and 50-nm suspensions were dispensed automatically via burst modules (Buehler). This protocol yields mirror-flat surfaces with ≤1 nm residual roughness, as previously verified by atomic force microscopy. When possible, we thinned the samples to 2 mm thickness, which is ideal for PEEM experiments. For the fossil samples (Vm, Eh, Hh, He and Pb) this was not possible; thus, we used a modified sample holder for the PEEM experiment. We trimmed the epoxy blocks to remove excess EpoFix resin and then cleaned them repeatedly with anhydrous ethanol and a new TexWipe Dry Cotton Cleanroom Wiper using gentle force until clean under differential interference contrast (DIC) microscopy (×20 objective). This step is key to obtaining perfect surfaces for the surface-sensitive PEEM method.
We then transferred all samples to ultrahigh vacuum (approximately 10−8 mbar) to remove any residual water. Several samples took many hours to outgas: we attempted the PA sample during 3 beamtimes over 2 years and it failed until we built a dedicated ultrahigh-vacuum chamber, called the Dalì chamber (Supplementary Fig. 1). The ultrahigh-vacuum chamber included a 660 l s−1 turbo pump ceramic-bearing turbomolecular Agilent Twistorr 704 (Agilent Technologies). The pump is mounted at the bottom of the Dalì chamber, equipped with a fast-entry flange on a side, and a drawer that comes out to receive the sample, and goes back into the chamber directly above the turbo, where the sample sits while pumping down.
We coated all samples twice using a Cressington 208HR (Cressington, Ted Pella) sputter coater. First, we coated the samples with 40 nm platinum and a 4 mm × 4 mm silicon wafer mask (MTI) to cover the functional cusp of the tooth and expose everything else to platinum, with no motion of the sample stage. Then, we removed the silicon mask and coated the whole sample with 1 nm platinum while spinning and tilting it. This coating method makes it possible to analyse with photoemission spectroscopy and microscopy any insulating sample including minerals60, biominerals61,62 and most importantly enamel22,63,64,65,66. At the calcium L edge, the maximum probing depth of the PEEM experiment is 3 nm (ref. 67); hence, a 1-nm coating enables much of the signal to originate from the sample under investigation. The 40 nm surrounding coating ensures good electrical contact between sample and sample holder, so that the sample can float at −18 kV without arcs and sparks, and the photoelectrons it emits can be accelerated towards the electrostatic optics column.
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