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

Area of Science:

• Molecular Biology
• Genomics
• Biotechnology

Background:

• Understanding cellular phenotypes requires preserving spatial context within intact tissues.
• Existing methods struggle to capture detailed molecular information in three-dimensional specimens.

Purpose of the Study:

• To introduce cross-linked volumetric DNA microscopy (xVDM) for high-resolution 3D molecular phenotyping.
• To demonstrate xVDM's capability in reconstructing cell-scale molecular communities within intact tissues.

Main Methods:

• xVDM involves seeding unique molecular identifiers into the tissue's protein matrix, linked by DNA bridges to form a proximity network.
• This network is used to reconstruct cell-scale molecular communities.
• The method was applied to zebrafish embryos and human tonsil tissue, utilizing antibody-oligonucleotide conjugates for protein targets.

Main Results:

• xVDM generates denser molecular networks and achieves broader transcriptome recovery compared to transcript-only methods.
• The technique successfully mapped genetically annotated 3D networks in zebrafish embryos at various developmental stages.
• Protein targets were also analyzed in human tonsil tissue, showcasing versatility.

Conclusions:

• xVDM offers a novel approach for 3D molecular phenotyping in intact specimens.
• The method relies on standard laboratory reagents and DNA sequencing.
• xVDM provides a powerful tool for studying cellular states and tissue organization in their native context.