Cross-linked volumetric DNA microscopy for dense molecular-network phenotyping in intact tissue

Resolving cellular phenotypes in full tissue context requires methods that can retain those cells’ physical neighborhoods, together with the identities of individual biomolecules, in intact three-dimensional specimens. We introduce cross-linked volumetric DNA microscopy (xVDM), in which unique molecular identifiers are seeded directly into the tissue’s protein matrix and linked by uniquely labeled DNA bridges to create a dense, DNA-encoded proximity network. Cell-scale molecular communities are then reconstructed directly from this network. xVDM produces denser molecular networks and broader transcriptome recovery than when these networks are nucleated by transcripts alone. xVDM maps out genetically annotated three-dimensional networks that map onto cell states and tissue regions in intact zebrafish embryos at 12, 18, and 24 hpf. Antibody-oligonucleotide conjugates extend the same framework to protein targets in human tonsil. xVDM provides a route to three-dimensional molecular phenotyping in intact specimens using only standard bench reagents and a DNA sequencer.