Ito slide

Fourty microns-thick transverse frozen sections were cut using a cryostat (Leica, Milton Keynes, UK) and fixed on a carbon-conductive adhesive tape which was in turn fixed on an indium tin oxide (ITO) slide (Bruker Daltonics, Bremen, Germany, cat no 237001).
All MSI measurements were performed using an Autoflex-Speed MALDI-TOF/TOF spectrometer (Bruker Daltonics, Bremen, Germany) equipped with a Smartbeam laser (355 nm, 1000 Hz) and controlled using the Flex Control 3.4 software package. The mass spectrometer was operated with a negative polarity in the reflectron mode. Spectra were acquired in the mass range of m/z 100–600 for all (x, y) coordinates corresponding to the imaged tissue.
The laser raster size was set at 50 microns. The signal was initially optimized by manually adjusting the laser power and the number of laser shots fired. Accordingly, full-scan MS experiments were run by accumulating 400 satisfactory laser shots per raster position, and using the laser power leading to the best signal-to-noise ratio. Image acquisition was performed using the Flex Imaging 4.0 (Bruker Daltonics) software package. The correlation of the target plate with the optical image was performed from three distinct teaching points following the procedure of the Flex Imaging software (Bruker Daltonics).

Samples preparation and data acquisition were performed under the same conditions as previously described by Gadea et al. [21 (link)]. Briefly, Pseudocyphelaria crocata samples were hand-cut using a razor blade to afford slices approximately 100 μm thick. Lichen slices were fixed on a carbon-conductive adhesive tape that was, itself, fixed on an indium tin oxide (ITO) slide (Bruker Daltonics, Bremen, Germany, cat. no. 237001). All MSI measurements were performed using an Autoflex-Speed MALDI-TOF/TOF spectrometer (Bruker Daltonics, Bremen, Germany) equipped with a Smartbeam laser (355 nm, 1000 Hz) and controlled using the Flex Control 3.4 software package. The mass spectrometer was used in the reflectron mode with a negative polarity. Spectra were acquired in the mass range of m/z 100 to 1000 for all (x, y) coordinates corresponding to the imaged tissue. The laser raster size was set at microns. The signal was initially optimized by manually adjusting the laser power and the number of laser shots fired. Accordingly, full-scan MS experiments were run by accumulating 400 laser shots per raster position and by using the laser power leading to the best signal-to-noise ratio. Image acquisition was performed using the Flex Imaging 4.0 (Bruker Daltonics) software package.

From each TMA, a section of 4 μm was adhered to an indium-tin-oxide (ITO) slide (Bruker Daltonics, Bremen, Germany). Sample preparation has previously been described in detail [15 (link),16 (link)]. Briefly, sample slides were heated to 80 °C prior to dewaxing with xylene (Carl Roth GmbH, Karlsruhe, Germany), and subsequent rehydration with increasingly concentrated ethanol washes (Carl Roth GmbH, Karlsruhe, Germany). Afterward, the samples were subjected to heat-induced antigen retrieval in MilliQ water at 95 °C for 20 min. A trypsin (Promega, Mannheim, Germany) solution was prepared in 40 mM ammonium bicarbonate (Sigma-Aldrich Chemie GmbH, Munich, Germany) to a final concentration of 0.1 µg/µL. The enzyme solution was sprayed with an automatic sprayer (TM Sprayer, HTX Technologies, Chapel Hill, NC, USA) in 16 cycles with a fixed spraying flow of 150 μL/min. On-tissue digestion was carried out for 2 h at a controlled temperature of 50 °C. Following digestion, four cycles of matrix solution (10 mg/mL of alpha-cyano-4-hydroxycinnamic acid matrix (Sigma-Aldrich Chemie GmbH, Munich, Germany) in 70% acetonitrile aqueous solution with 1% trifluoracetic acid (Carl Roth GmbH, Karlsruhe, Germany)) were deposited with a defined flow of 120 μL/min and a temperature of 75 °C.