INSIHGT: Accessible multimodal systems biology with quantitative molecular phenotyping in 3D

Biological systems are complex, encompassing intertwined spatial, molecular and functional features. However, methodological constraints always limit the completeness of information that can be extracted. Here, we report the development of INSIHGT, a minimally perturbative, accessible and cost-efficient three-dimensional (3D) spatial biology method utilizing superchaotropes and host-guest chemistry. This allows highly multiplexed and multi-modal readout of tissue biomolecules in biological systems up to centimeter scales, permitting radio-histological correlation of phosphorylated alpha-synuclein pathologies in human hemi-brainstem. The homogeneous penetration permits reliable semi-quantitative signals in 3D compared to reference signals. Diverse antigens, mRNA transcripts, neurotransmitters, and post-translational and epigenetic modifications, are well-preserved and visualized. INSIHGT also allows multi-round molecular probing for high-dimensional spatial biology and compatibility with downstream traditional histology. With INSIHGT, we mapped previously undescribed podocyte-to-parietal epithelial cell microfilaments and demonstrated their geodesic clustering in mouse glomeruli, and catalogued sparsely located neurofilament-intensive inclusion bodies in the human cerebellum, and identified NPY-proximal cell types defined by spatial morpho-proteomics in mouse hypothalamus. We anticipate INSIHGT can form the foundations for 3D spatial multi-omics technology development and holistic systems biology studies.