Multimodal Imaging of the Cellular and Extracellular Microenvironment on the Same Formalin-Fixed Paraffin-Embedded Tissue Section.

Same-tissue section multimodal imaging is a powerful strategy that spatially profiles tissue histology, cell populations and molecular composition while maximizing tissue economy, preserving spatial molecular relationships, and increasing co-registration capacity. However, performing multiple modalities on the same tissue section can destroy or chemically alter the tissue, compromising downstream data. Here, we systematically assess integration of picrosirius red staining, hematoxylin and eosin staining, and multiplexed immunofluorescence into N-glycan and extracellular matrix peptide matrix-assisted laser/desorption ionization imaging mass spectrometry (IMS) workflows. We evaluate alterations in tissue morphology, stain efficiency, IMS feature intensity as well as IMS feature localization after upstream modality integration. We propose an optimized multimodal sequence that maximizes data quality and follows a very specific order of: autofluorescence microscopy, multiplexed immunofluorescence, picrosirius red staining, N-glycan IMS, hematoxylin and eosin staining, and extracellular matrix peptide IMS. Overall, this work develops an optimized multimodal workflow that comprehensively images tissue morphology, collagen fibers, and cell populations at single-cell resolution as well as multiplexed N-glycan composition and multiplexed extracellular matrix peptides with post-translational modification status from a single tissue section.