Recent improvements in electron microscopy have had a profound impact on cellular biology as they promise to bring a long-held 'holy grail' within reach: the ability to directly visualize nanoscale processes occurring within a cell. This goal—in situ visualization—is important because biological components often change their structure or function when removed from their native cellular environment. This grant supports a project by a team of five principal investigators to build a new type of microscope—a cryo-super-resolution microscope optimized for correlative light- and electron-microscopy (CLEM) that produces 3D snapshots (cryo-electron tomography; cryo-ET)—and to demonstrate the new microscope’s usefulness for the in situ visualization of intracellular structures and processes. Grant funds will support microscope design, construction of the super-resolution microscope, creation of software to run it, and the development of “workflows” defining how to implement CLEM when cryo-ET is the electron-microscopy technique of interest. Additionally, the principal investigators will conduct four demonstration projects in biology that leverage the capacities of the new microscope to obtain in situ structural explanations of a major cellular process. The first project, the clathrin project, seeks to understand force generation during clathrin-mediated endocytosis (CME), the taking-in of matter by invagination of a cell membrane. The second will produce images that improve our understanding of autophagy, the engulfment and degradation of various harmful objects within a cell. The third project seeks to understand the basic mechanisms behind the formation of a primary cilium, a hair-like entity that serves as an important sensory organelle on virtually all animal cells. The fourth project will use microscopy to visualize (polycomb-regulated) changes in chromatin structure during cellular development. The proposed work represents a next-logical-step in the development of electron-microscopy-for-biology and our corresponding ability to study the cell, the most basic unit of life. To help push this emerging technology out to a broad community, information on how to build and use the microscope will be made publicly available and access to the new microscope will be provided to scientists from across the globe through a visitor program.