Project Summary/Abstract Adaptive immune response, brain development, and tumor progression all display physiology that depends on somatic mutations and genomic rearrangements among interacting cells. There is currently no widely-available tool to capture nucleotide-level variation in a tissue’s three- dimensional context. In my lab, we are developing spatial-genetic technologies to bridge this major technological gap. DNA microscopy, an imaging modality that I developed for generating spatial-genetic maps of tissue de novo (Weinstein, Regev, Zhang, Cell 2019) provides a critical foundation for this work. DNA microscopy encodes the spatial positions and nucleotide-level differences of cells into the DNA products of a stand-alone chemical reaction. The technology operates by first tagging individual DNA and RNA molecules with unique DNA barcodes, and then turning these barcoded molecules into an intercommunicating network: linking barcodes to one another at rates that depend on the distance between the original molecules. In doing so, DNA microscopy uses DNA as an imaging medium, effectively imaging a specimen from the “inside-out”. My lab’s research has two thrusts. The first of these is aimed at exploiting the fact that DNA microscopy image-capture is intrinsically volumetric, and applying it to deep-tissue three- dimensional spatial-genetic imaging. The second thrust of our research is aimed at using the phylogenetic relationships encoded into the DNA of proliferating cells, via genomic mutations and other forms of stochastic genomic reorganization, in order to ultimately decode from their genomes information about cellular dynamics through time. Our goal is to use DNA microscopy’s capability to jointly resolve cellular positions and cell clonal relationships to reconstruct their spatial and temporal dynamics in model organism development. We further aim to apply this framework to deepen our understanding of genomic variability in tumors and immune cells in mammalian tissue. In this proposal, I describe our plan to achieve both of these goals, and to establish a foundation for DNA microscopy to be deployed as a critical tool for spatial-genetic imaging in both basic biology and pathology.