1. Fix sample on MALDI target holder with superglue, perform 3D optical microscopy. 2. Dissolve DHB (30 mg/ml) and CHCA (7 mg/ml) in 1 ml 1:1 acetone/water with 0.1% TFA. 3. Apply 100 µl matrix solution using a pneumatic sprayer system at 10 µl/min and 1 bar. 4. Check matrix layer quality with an optical microscope. 5. Calibrate mass spectrometer with known DHB cluster signals. 6. Mount MALDI target with sample on the target support of AP-SMALDI10 MSI source. 7. Search region of interest in the optical sample observation camera. 8. Set mass spectrometer to m/z 100-1000, fixed injection time of 500 ms, and apply 4 kV to the MALDI target. 9. Set step size (10-30 µm) and pixel number in the control software. Adjust laser pulse energy. 10. Set lock mass to calibrate spectra of each pixel to known matrix cluster peaks. 11. Start mass spectrometer data acquisition and initialize 3D scan. 12. Load data in MIRION or MATLAB software. 13. Set image bin width to m/z ±0.01. 14. Export relevant images as .jpg and .csv files. 15. Use 3D imaging data to display sample topography in heat map color scale using MATLAB. 16. Combine three ion images into an RGB overlay image and integrate with topographic information into 3D RGB MS surface images. 17. Normalize MS images to the base pixel per image. 18. Import m/z value list into METLIN database for accurate mass compound assignment.