Fig 2: MALDI MSI images of fresh-frozen tissues (left image) compared with FFPE tissue samples (right) using the optimized SOP. Shown are MALDI MSI images of a mouse kidney using the optimized SOP and overlaid on the tissue scan. 718 (m/z) green, 958 (m/z) blue (a, b accumulated by using an Ultraflex mass spectrometer; c accumulated by using Rapiflex mass spectrometer (both from Bruker Daltonics, Bremen, Germany)). a Partial resolution 50 µm. b Partial resolution 25 µm. c Partial resolution 10 µm

Fig 3: Lipid-protein GAMSI.Spatial distributions of target protein peaks and major lipid peaks from expanded mouse cerebellum (PFA-fixed). The instrument pixel size (Bruker rapifleX) was set at 50 µm. Scale bars: 200 µm (760 µm). DG: diacylglycerol; PS: phosphatidylserine; PA: phosphatidic acids; PE: phosphatidylethanolamine; PG: phosphatidylglycerol; PI: phosphatidylinositol; SHexCer: sulfatides; HexCer: hexosylceramides; ST: sterols; TG: triglyceride.

Fig 4: IMS images of LMW fragments specific or highly enriched in cataract lenses. Images were acquired on Bruker Rapiflex Tissuetyper using a 150 µm raster step size. Signal represents peak m/z value ± 0.01% m/z unit.

Fig 5: (A). Key steps in MALDI-IHC. Photocleavable Mass-Tag (PC-MT) labeled probes, including antibodies and lectins, are used for high-plex tissue imaging. An additional initial step (not shown) can be performed prior to MALDI-IHC to image endogenous small molecules such as metabolites and drugs (See main text for details). (B). Schematic of a dual-labeled Miralys™ antibody probe with a photocleavable peptide mass-tag and fluorophore conjugated to the antibody. Note the diagram does not reflect the actual attachment sites of the peptide mass-tag or fluorophores, or the actual number of tags conjugated to the antibody. (C). Example MALDI-IHC workflow. Both FFPE and FF tissue samples can be imaged using MALDI-IHC. Staining utilizes Miralys™ probes which are photocleaved prior to matrix deposition, MALDI-MSI and analysis. Up to 10 slides can be automatically stained in 10 h using the AmberGen automated stainer. At 40 µm resolution a 1 cm2 tissue specimen can be imaged on a Bruker rapifleX or timsTOF fleX instrument in less than 1 h.