TOF-SIMS (Michellod et al. 2023)

PFA-fixed Olavius algarvensis specimens were embedded in paraffin and cut into 4 µm thick sections. The sections were de-waxed, wrap in aluminium foil and stored in a dessicator until TOF-SIMS analysis. SIMS data were acquired on an IONTOF TOF-SIMS 5 instrument (IONTOF GmbH) using a 25 keV Bi3+ LMIG analytical beam in positive mode. To obtain high-resolution mass spectra, high current bunch mode was used with a beam current of ~1 pA. The analytical area was typically 100 µm2. Secondary ion maps were collected using the Bi3+ LMIG in burst alignment mode for greater lateral resolving power (raster size: 512 x 512, FoV 210 x 210). The samples were also sputter pre-cleaned using a 5 keV Ar1000+ cluster ion beam. We analyzed standards to determine the most abundant ions produced by each sterol: cholesterol ([M-H2O+H]+ C27H45, at m/z 369.38; [M-H]- C27H45O, at m/z 385.34; [M-H+O]- C27H45O2, at m/z 401.35) and sitosterol ([M-H2O+H]+ C29H49, at m/z 397.47; [M-CH3O]- C28H47, at m/z 383.37; [M-H]- C29H49O, at m/z 413.45).