Project Summary/Abstract Organ Specific Project Component Multi-Scale Multi-Modality High Resolution Mapping of the Adult Human Lung Maintaining normal lung function and inducing repair of damaged lung can be markedly improved if we better understand the cellular and molecular complexities of the organ. Technical and analytical advances now make it possible to gain detailed access to previously hidden fundamentals of lung biology by generating high resolution, single cell and subcellular, biomolecular maps coalesced into an atlas of the human respiratory system. It is the goal of HuBMAP and specifically of this Lung TMC to generate interactive, searchable, cell type and subtype annotated maps and tabular data representing transcriptome, proteome, metabolome, and select posttranslational modifications (PTMs) of healthy lung. Ultimate deliverable will be integration of these maps into a reference atlas of healthy lung to which diseased lung can be compared. Such a biomolecular atlas with multi- scale, 3D spatial resolution will be truly transformative for human pulmonary biology with ramifications also for understanding the immune and vascular systems throughout the body. Building upon our successful HuBMAP Phase 1 engagement, our tissue mapping strategy brings multiple strengths including 1) an established, robust, tissue biorepository, 2) well oriented tissue samples representing the entire human lung with focus on regions of interest, 3) snRNAseq/ATACseq dual-omic sequencing and mass spectroscopy based bulk metabolomics to provide basis for deconvolution of, 4) novel spatial transcriptomic methods, 5) spatial highly-multiplexed chemical and immunofluorescent microscopy, 5) a suite of unbiased mass spectrometry, stimulated Raman spectroscopy and advanced microscopy technologies, all combined in such a way to enable spatially resolved co-localized 2D and 3D mapping of gene expression, metabolites, lipids, proteins, and protein modifications across lung tissues with single-cell or near single-cell resolution. We will include assays on 40 diverse donor lungs creating tissue microarrays to enhance assay throughput. We envision achieving an unprecedented level of biomolecular mapping of the trachea, extra and intra-pulmonary bronchi with bronchial artery and lymphatic systems, several levels of bronchioles with focus on broncho-vascular bundles and terminal-respiratory-alveolar duct transition regions as well as alveolar and pleural regions. Working with the HuBMAP HIVE and our DAC, our achievable goal will be, by microdissection, serial section reconstruction and 3D imaging of thick sections, a Multi-Scale Multimodality High Resolution 3-D and Tabular Atlas of the Adult Human Lung.