PROJECT ABSTRACT Our brain is the most complex system under study. It relies on precise wiring and signaling across billions of neurons. Our neurons span centimeters, communicating with one another via a sea of sub-micrometer synapses and nanoscale proteins. At the same time, each of our neurons has a distinct type, stemming from its unique expression of thousands of genes, and can assemble into spatial gradients with other neurons that span entire regions of our brain. Charting vast multi-dimensional maps of our brain is one of the greatest challenges of modern neuroscience, with far-reaching implications for shedding light on the mechanisms of brain function and improving our ability to diagnose and treat neurological diseases. However, the size and scale of these maps is unprecedented, as they require us to see the big (i.e., the entire brain) and the small (i.e., individual synapses, proteins, and RNA transcripts) in a highly multiplexed manner (i.e., many molecular markers). A growing number of recent innovations in tissue processing, including tissue expansion for microscopy (physically enlarging tissue), multiplexed antibody labeling (protein mapping), and in situ sequencing (reading out mRNA in tissue using sequencing by synthesis), now make it possible to visualize hundreds of molecular entities in three dimensions across centimeter-scale tissue volumes at resolutions that exceed the diffraction limit. At the same time, the cost of data storage and computation continues to decrease according to Moore’s law. These breakthroughs open the door to a new frontier of scientific discovery that is not bounded by our ability to interrogate the molecular contents of tissue, or store and process the resulting imaging data. This frontier is instead severely bounded by our current imaging technologies and the rate at which we can collect high- resolution, highly multiplexed data from large tissue volumes. Here I propose to develop a pair of Exa-scale Tissue Readout Methods (ExTReMe) that overcome this boundary and improve imaging throughput by orders of magnitude over current state-of-the-art approaches. I will develop a first ExTReMe platform to enable a new type of molecular interrogation of neural circuits, mapping individual proteins at molecular resolutions, in a highly multiplexed manner, across entire brains. I will develop a second ExTReMe platform to scale in situ sequencing methods to larger mammalian brains. Mapping neuron types across the macaque or human brain will be accomplished in several months or years, as opposed to several decades or centuries. These new imaging tools will transform our understanding of the brain’s cell types and their connections and provide detailed molecular fingerprints of neurological and neuropsychiatric disorders. Although this project focuses on applications in brain research, both ExTReMe platforms will have broad impact in other fields, including but not limited to oncology, immunology, and developmental...