A. MALDI-MSI of heat fixed tissue after pathogen inactivation. Tissue sections (10 µm) were prepared for MALDI-MSI of proteins and peptides by spraying with sinapinic acid. a) H&E stained spleen serial section and pseudocolored MALDI-MSI ion overlay showing the expression pattern of m/z 8416 (green: located in areas of periarteriolar lymphocyte sheath), and m/z 14986 (red: surrounding tissue). b) H&E stained serial lung tissue section and pseudocolored image overlay showing the expression pattern of m/z 12167 (red: located in the primary bronchus), and m/z 13800 (green: surrounding tissue). All masses are reported with a mass window of 0.4%. B. Overlay of representative MALDI-MSI spectra (500 combined laser shots) obtained from corresponding regions of heat fixed (red trace) and snap frozen (black trace) mouse brain tissue. Arrows indicate the ions mapped in the inset image. Inset- pseudocolored ion image (top) and H&E of an adjacent section (bottom) of a heat fixed mouse brain section showing the distribution of m/z 14121 (red) and m/z 6688 (green) located in two regions of the midbrain. C. MALDI-MSI of heat fixed and snap frozen mouse brain tissue for lipid detection. (a) Average total MALDI spectra in reflector negative ionization mode obtained from heat fixed (HF) and snap frozen (SF) mouse brain display numerous peaks representing lipid ions in the m/z 700-950 region. (b-c) Scanned images of H&E stained and matrix (DHB) sprayed serial sections (left 2 panels) and pseudocolored overlays of ion images (right panels) in heat fixed (HF) (b) and snap frozen (SF) (c) mouse brain. Horizontal mouse brain sections showing the expression pattern of two lipid species: m/z 885.6 (green) in the inferior and superior colliculus and m/z 715.5 (red) in the cerebral cortex. Dashed lines indicate MALDI-MSI measurement regions. All masses are reported with a mass window of 0.1%. IC- inferior colliculus, SC-superior colliculus, CC- cerebral cortex, Cb-cerebellum.