Surfacelab 7

Peak assignments for each sample were created by IonTOF SurfaceLab 7. Peak lists of secondary mass ions and secondary intensity from the software were exported and then imported into the LIPIDMAPS database to identify the lipid species. For metabolite results, 3D OrbiSIMS spectra were exported as .txt files and then searched against the Human Metabolome Database with a 5 ppm mass tolerance for putative annotation.

TOF-SIMS data
analysis was performed using SurfaceLab 7 (IONTOF GmbH, Germany).
MALDI-MSI data were analyzed using Thermo Xcalibur 2.2 and Thermo
ImageQuest (Thermo-Fisher Scientific, USA), METASPACE (https://metaspace2020.eu), and
LipostarMSI (Molecular Horizons Srl, Italy). All images were normalized
to total ion count (TIC) and denoised by hotspot removal. Segmentation
(bisecting K-means algorithm), colocalization, and PCA analyses were
performed using LipostarMSI.

Mass spectrometry imaging was performed using a HybridSIMS instrument (ION-TOF GmbH) employing a 30 keV Bi₃⁺ primary ion source (0.3 pA target current). In order to best accommodate sample topography, delayed extraction was used for analysis with nitrogen gas flooding. Data was acquired over a 200 × 200 µm² area, with a resolution of 256 × 256 pixels with 20 shots per pixel. The data acquisition and analysis was performed with SurfaceLab7 (ION-TOF GmbH). Charge neutralisation was performed using a relatively low energy (< 2 eV) electron floodgun.

ToF-SIMS data were acquired using a ToF-SIMS IV instrument (ION-TOF GmbH., Münster, Germany). The primary ion beam was a bismuth liquid metal ion gun (Bi3+) run at 25 kV (pulsed target current of ~1 pA). For each sample, data were acquired over 3 regions of 500 µm × 500 µm at 256 × 256 pixels resolution for 20 scans. Data acquisition and analysis was performed using SurfaceLab 7 software (IONTOF GmbH). Selected spectra (area 1 of each sample) were normalised for comparison and identical Y scales were used.

3D OrbiSIMS mass spectrometry imaging was performed using a HybridSIMS instrument (ION‐TOF GmbH) employing a 30 keV Bi₃⁺ primary ion source (0.3 pA target current) in delayed extraction mode to analyze the AMS samples. Data was acquired over a 4 × 4 mm area, with a resolution of 100 pixels per mm with 15 shots per pixel. The data acquisition and analysis was performed with SurfaceLab7 (ION‐TOF GmbH). Charge neutralization was performed using a relatively low energy (<2 eV) electron floodgun.

Analysis of the printed proteins before and after washing with DI water was conducted using a ToF–SIMS IV instrument (IONTOF GmbH, Münster, Germany) with a Bi³⁺ cluster primary ion source operated at 25 kV. The primary ion dose density was maintained at < 1 × 10¹² ions/cm² to ensure static conditions. Positive polarity spectra were acquired in high current bunched mode over 1 × 1 mm areas at a resolution of 228 pixels per mm, using the macroraster stage function. Retrospective data analysis allowed ions indicative of amino acids (e.g., CH₄N⁺, C₂H₆N⁺, C₄H₈N⁺) to be identified. Positive spectra were mass calibrated using CH₃⁺ (m/z 15), C₂H₅⁺ (m/z 29), C₃H₇⁺ (m/z 43), and C₄H₉⁺ (m/z 57) peaks. Data analysis was carried out using SurfaceLab 7 software (IONTOF GmbH, Münster, Germany).

Material samples were cut into 1 cm² coupons and measured by ToF–SIMS as received from manufacturer without additional processing. ToF–SIMS data were collected using a ToF–SIMS IV instrument (ION-TOF GmbH., Münster, Germany) equipped with a bismuth liquid metal ion gun and a single-stage reflectron analyser. Bi₃⁺ primary ion energy of 25 kV and a pulsed target current of approximately 1.3 pA were used in this measurement. Low-energy electrons (20 eV) were used to compensate for surface charging due to the positively charged primary ion beam on the insulating surfaces. Rastered areas of 3 × 3 mm² were analysed at a resolution of 100 pixels per mm and 15 frames per patch. The total primary ion beam dose for each analysed area was kept below 1 × 10¹² ions per cm², ensuring static SIMS acquisition conditions. Data acquisition and analysis were performed using IONTOF SurfaceLab7 software (IONTOF, Münster, Germany).