Fig 2: (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.

Fig 3: Multimodal high-resolution MALDI-IHC imaging of FFPE breast cancer tissue. (A) Two-color immunofluorescence microscopy image using dual-labeled Miralys™ probes (Na+/K+ ATPase and histone H2A.X, yellow and red, respectively). (B) MALDI-IHC image at 5 µm pixel size using Bruker timsTOF fleX equipped with the microGRID accessory to image the mass-tags from the same dual-labeled probes (Na+/K+ ATPase and histone H2A.X, blue and red, respectively). (C) Merge of the fluorescent and MALDI-IHC images shown in (A) and (B) for a selected region (see cross-hatched yellow rectangle in (A). The MALDI-IHC image comprised of 5 × 5 µm pixels for histone H2A.X (in this case the square yellow pixels) was interactively aligned with the fluorescent image comprised of individual cell nuclei colorized as red, also histone H2A.X.