Figure 1: Negative ion ToF-SIMS spectra of melanin and microbodies from FUM-N-2050.

(a) Optical photograph of the specimen. Scale bar: 10 mm. (b) Close-up of brownish matter located in the orbit; position of the area analysed by SEM, TEM, ToF-SIMS and IR microspectroscopy indicated by an arrow. Scale bar: 1 mm. (c) Detail of the eye (SEM image) showing closely spaced, elongate and oblate melanosome-like structures preserved as solid bodies. Scale bar: 2 μm. (d) A semi-transparent ion image showing the distribution of melanin-derived ions (identified from natural and synthetic melanin standard spectra; Table 1; Supplementary Fig. S3) superimposed onto a SEM image of tightly packed melanosome-like bodies. The added signal intensity from C₃N⁻ (m/z 50), C₃NO⁻ (m/z 66) and C₅N⁻ (m/z 74) is shown in orange-yellow, whereas areas high in signal from peaks at m/z 145 and m/z 146 are shown in green (see Table 1 for assignments). The purple line demarcates the area from which the mass spectrum presented in (e) ('fossil fish eye') was collected. Scale bar: 2 μm. (e) Negative ToF-SIMS spectra representing the eye of FUM-N-2050 and a natural melanin standard (from Sepia officinalis). The fossil fish eye spectrum was reconstructed from a ~4 μm² area in a measurement at 50×50 μm² acquired at high spatial resolution (mass resolution m/Δm ~300) to allow for analysis of ToF-SIMS data specifically from within the selected area marked in (d), whereas the melanin standard spectrum was recorded at high mass resolution (m/Δm ~5000). The close agreement between the two spectra (both with regard to their peak positions and relative intensity distributions) provides compelling evidence for a high melanin content in the eye of FUM-N-2050. Filled circles indicate peaks used to produce the ion image in (d), whereas crosses indicate peaks from inorganic ions that are not part of the melanin structure (and not present in the synthetic melanin standard spectrum; Supplementary Fig. S3).