Mirax desk slide scanner

MSI was performed in positive ion reflector mode over a mass range of m/z 600–3200 on rapifleX MALDI Tissuetyper TOF mass spectrometers (Bruker) fitted with a Smartbeam 3D Nd:YAG (355mm) laser. The Smartbeam parameter was set to M5 small, with the Imaging 50 µm application, 15 µm scan range, and resulting field size of 50 µm. Spectra were accumulated from 500 laser shots at 10 kHz frequency with a sampling rate of 1.25 GS s^–1 and baseline subtraction performed during acquisition. The “Detector check” function was conducted regularly at each site in order to maintain this variable setting at appropriate voltages. For every measurement, the instruments were externally calibrated using the Peptide Calibration Standard II (Bruker),12 laser power adjusted according to on‐tissue test shots in order to reach the optimal ionization threshold, and two non‐tissue measurement regions included. Following MALDI–MSI, the matrix was eluted from the samples with 70% ethanol, stained with hematoxylin and eosin (H&E) and scanned with 20× objective magnification on a NanoZoomer SQ (Hamamatsu Photonics Deutschland, Munich, Germany), Aperio AT (Leica Biosystems, Buffalo Grove, IL, USA), or Mirax Desk slide scanner (Carl Zeiss MicroImaging, Göttingen, Germany). The H&E image was then co‐registered to the respective measurement in flexImaging v. 5.0 or SCiLS Lab MVS 2018b software (Bruker).