349 nm laser

Tissue sections of 12 μm thickness were thaw-mounted onto poly-l-lysine-coated (Sigma-Aldrich, Saint Louis, MO, USA) indium-tin oxide (ITO) conductive slides (Bruker Daltonics, Billerica, MA, USA) and stored at − 80 °C until use. ITO slides were thawed under vacuum for 15 min, and 8 layers (2 layers at 5 µl/min followed by 6 layers at 10 µl/min) of 2,5-dihydroxybenzoic acid (DHB (Sigma-Aldrich, Saint Louis, MO, USA) 30 mg/ml in MeOH:H₂O 70:30, 0.2% TFA) were applied using a SunCollect (SunChrom, Friedrichsdorf, Germany) automated matrix spraying system (4 bar, 50 mm z axis height). The tissue sections were then dried under vacuum for 15 min prior to MALDI MSI data acquisition. MALDI MSI was performed with an EP-MALDI source [27 (link)] (Spectroglyph, LLC., Kennewick, WA, USA) equipped with a 349-nm laser (Spectra-Physics, Santa Clara, CA, USA), coupled with an Orbitrap QExactive Plus (Thermo Fisher Scientific). The laser was operated at 1.65 A and 500 Hz, the ion source pressure was 7.2 Torr, and the MSI pixel size was 35 × 35 µm. Mass spectra were acquired in the range 150–2000 m/z at 70,000 resolving power. The position file was aligned to the raw file using Image Insight (v. 0.1.0.11550, Spectroglyph, LLC).

Tissue sections mounted on ITO glass slides were thawed under vacuum for 15 min prior to matrix application. One replicate was processed for each condition. The MALDI matrix norharmane (7 mg/mL in CHCl₃:MeOH 70:30) was sprayed onto the tissue sections using a SunCollect automated spraying system (SunChrom, Friedrichsdorf, Germany). X and Y deposition speed were set to Low 3 and Medium 1 respectively while the height of the nozzle was set to 50 mm. The matrix solution was applied in 8 layers (2 layers at 5 µL/min followed by 6 layers at 10 µL/min). The matrix coated tissue section was then dried under vacuum for 15 min prior to data acquisition.
MALDI MSI was performed using an elevated pressure (EP) MALDI source^{20 (link)} (Spectroglyph, LLC., Kennewick, WA, USA) equipped with a 349 nm laser (Spectra-Physics, Santa Clara, CA, USA), coupled to an Orbitrap Q-Exactive Plus mass spectrometer (Thermo Scientific). The laser was operated at 1.65 A and 500 Hz, the source pressure was 7.2 Torr and the pixel size was 30 × 30 µm. Mass spectra were acquired in the range 150–2000 m/z at 70 000 resolving power at m/z 200. The position file was aligned to the raw MS files using ImageInsight (v. 0.1.0.11550, Spectroglyph, LLC).

Tissue sections of 12 μm thickness were thaw-mounted onto poly-l-lysine-coated (Sigma-Aldrich, Saint Louis, MO, USA) indium-tin oxide (ITO) conductive slides (Bruker Daltonics, Billerica, MA, USA) and stored at − 80 °C until use. ITO slides were thawed under vacuum for 15 min, and 8 layers (2 layers at 5 µl/min followed by 6 layers at 10 µl/min) of 2,5-dihydroxybenzoic acid (DHB (Sigma-Aldrich, Saint Louis, MO, USA) 30 mg/ml in MeOH:H₂O 70:30, 0.2% TFA) were applied using a SunCollect (SunChrom, Friedrichsdorf, Germany) automated matrix spraying system (4 bar, 50 mm z axis height). The tissue sections were then dried under vacuum for 15 min prior to MALDI MSI data acquisition. MALDI MSI was performed with an EP-MALDI source [27 (link)] (Spectroglyph, LLC., Kennewick, WA, USA) equipped with a 349-nm laser (Spectra-Physics, Santa Clara, CA, USA), coupled with an Orbitrap QExactive Plus (Thermo Fisher Scientific). The laser was operated at 1.65 A and 500 Hz, the ion source pressure was 7.2 Torr, and the MSI pixel size was 35 × 35 µm. Mass spectra were acquired in the range 150–2000 m/z at 70,000 resolving power. The position file was aligned to the raw file using Image Insight (v. 0.1.0.11550, Spectroglyph, LLC).