Pathogenic infection and survival within a host often rely on the exploitation of host lipids, yet we lack a basic understanding of how these scavenged molecules are transported and spatially organized to form a functional, protective cell envelope. The Lyme disease spirochete, Borrelia burgdorferi, is an ideal model to address this knowledge gap. B. burgdorferi relies entirely on the host environment to scavenge essential lipids to assemble its membranes and to produce glycolipids critical for pathogenesis. While lipidomic studies have identified the chemical composition of these stolen lipids, current imaging modalities like fluorescence microscopy and cryo-electron tomography lack the resolution and chemical specificity, respectively, to resolve their precise distribution within the cell envelope. To tackle this challenge, the Jacobs-Wagner and Dahlberg laboratories will collaborate to develop a multimodal imaging pipeline using element-labeled lipids and cryogenic electron tomography and electron spectroscopy. This novel approach will leverage the element-specific energy signatures of X-rays to distinguish scavenged lipids at nanometer-scale resolution. This will be the first direct visualization of how a pathogen coordinates its lipid landscape with full subcellular structural context. Beyond advancing our knowledge of B. burgdorferi architecture, this project will deliver a broadly generalizable, genetics-independent imaging toolkit applicable to a broad range of human pathogens, expanding our understanding of host-pathogen lipid transfer from the ‘what’ to the ‘where’.