Time-of-flight secondary ion mass spectrometry (ToF-SIMS)

ToF-SIMS is a surface analysis technique that provides spatially resolved molecular information from solid materials. During examination, a focussed beam of high-energy (primary) ions bombards the sample and mass spectra of the emitted (secondary) ions are recorded⁶⁶. Ion images showing the spatial distribution of specific molecular species and mass spectra from selected structures are generated by scanning the primary ion beam across the sample surface.

In this study, both untreated and demineralised fossil samples were fixed for ToF-SIMS analysis on silicon wafers using either double-sided tape or Milli-Q water. Additionally, natural eumelanin from the cephalopod Sepia officinalis (Sigma-Aldrich) and hemin (MP Biomedicals) were fixed on solid substrates using double-sided tape. The fossil data were compared also to a number of other standard compounds, including various pheo- and pyomelanins^20–22, hopanoids⁶⁷, peptidoglycans (from Micrococcus luteus and Methanobacterium sp., provided by Sigma-Aldrich), and porphyrins [chlorophyll a²⁰, protoporphyrin IX (Sigma-Aldrich), protoporphyrin X with copper (Sigma-Aldrich), uroporphyrin I dihydrochloride (Sigma-Aldrich), coproporphyrin I dihydrochloride²¹, and copper II phthalocyanine²¹].

ToF-SIMS analyses in the static SIMS mode were conducted in a TOFSIMS IV instrument (IONTOF GmbH) using 25 keV Bi₃ ⁺ primary ions and low energy electron flooding for charge compensation. Positive and negative ion data were acquired with the instrument optimised either for high mass resolution (m/∆m ~5,000, spatial resolution ~3–4 µm) or high image resolution (m/∆m ~300, spatial resolution ~0.2–0.5 µm). The pulsed primary ion current was 0.10 pA for the high mass resolution data and 0.04 pA for the high image resolution data.