The complexity of the mammalian brain is unparalleled by any other organs, and understanding its cellular composition and their brain-wide organization is essential to understand the brain functions and dysfunctions. Extensive efforts have been made toward mapping brain cells through various lenses, and have established invaluable databases yielding new insights. However, only a few molecules, or cell-types, or regions per brain have been mapped in non-human primate and human brains, and fail to capture brain-wide complex intercellular relationships within individuals. We here aim to develop versatile and easy-to-adopt platform technologies for highly multiplexed, rapid, scalable proteomic mapping of mammalian brains from mouse, marmoset, to human. We propose to (Aim 1) develop molecular toolkits and platform technologies (ELAST-SABER) for highly multiplexed scalable proteomic phenotyping of mouse, marmoset, human brains. We will develop a library of orthogonal DNA-barcoded antibodies targeting over 100 canonical cell-type markers, compatible with SABER amplification (developed by the group of PI Peng Yin) and ELAST transport (developed by the group of PI Kwanghun Chung). We will also (Aim 2) validate the technology platform and demonstrate its broad utility and scalability by mapping the cellular landscape in the primary motor cortex (M1) of mouse, marmoset, human brains including tissues with amyotrophic lateral sclerosis (ALS) pathology. We will cross-validate our method using the published MERFISH and single-cell sequencing data from BICCN. We will perform cross-species comparative analysis to understand conserved or evolutionarily distinct cellular features of M1. Finally, we will (Aim 3) establish web-based interactive platforms for data dissemination and community use. We will build multimodal brain atlases allowing cross species comparison, and develop neuro-ontology of the mouse, marmoset and human brain. Importantly, we will develop a database built with informatics and online visualization tools that allow people to view, compare, and analyze multiplexed scalable proteomic imaging data across three species. These informatics tools will have the capacity to standardize and accommodate histological, connectivity, gene expression, and in vivo imaging. Users will be able to compare multi-modality data for the ?most comparable structures? either based on their neuroanatomic nomenclature, ontology relationship, similarity of their protein markers, or neural connectivity. These tools will become openly available to the community. Our technology platform will enable us to establish the most comprehensive 3D map of the primary motor cortex (M1) of marmoset and human brains including tissues with amyotrophic lateral sclerosis (ALS) pathology to date? providing a foundation for mapping cell types brain-wide in the future and for studying pathological changes in brain disorders. We envision that these scalable tools will empower the research community to interrogate brain structure and function, including complex intercellular relationships at multiple scales.