PROJECT SUMMARY ABSTRACT Ligand-triggered events are central to many processes in neuroscience, endocrinology, virology, immunology, and pharmacology. However, molecular and ultrastructural changes that follow the stimulus are difficult to visualize because they involve rapid nanoscale motions and modifications of proteins and membranes. State-of- the-art techniques are insufficient to capture these spatiotemporal changes. For example, live fluorescence imaging is limited by the spatial resolution (diffraction-limit) and labeling constraints (no antibody access or washing in live cells), while nanoscale imaging methods either lack temporal resolution to capture fast dynamics (e.g., super-resolution optical microscopy) or are incompatible with live-cell imaging altogether (e.g., standard or cryo-electron microscopy; expansion microscopy). Given these limitations, time-resolved cryo-vitrification methods are ideal for capturing cellular processes after a defined wait time post-stimulation by freezing samples in the state of amorphous ice prior to imaging. High-pressure freezing (HPF) is often used for this purpose because of its relaxed sample thickness constraints (<300 µm) as compared to cryo-plunging at atmospheric pressure (<10 µm). However, an HPF device compatible with time-resolved buffer exchange does not currently exist. To this end, we will develop HPF-X – an HPF device with a capability for time-resolved buffer exchange preceding cryo-vitrification. Buffer exchange will allow stimulating the sample with various biological and pharmacological agents including ions, small molecules, peptides and proteins (e.g., hormones, cytokines, antibodies, and nanobodies), and even viruses and cells. Thus, HPF-X will allow cryo-vitrifying cells, tissue samples, or entire small organisms at a series of time points following stimulation with ligands for subsequent interrogation with nanoscale imaging techniques such as electron microscopy and super-resolution optical microscopy. This approach will allow capturing ligand-triggered cellular processes with nanoscale spatial resolution and temporal resolution of <50 ms. Biological applications of this technique include nanoscale imaging of protein-protein interactions, post-translational modifications, and protein-membrane dynamics. Although a fundamentally new HPF instrument design is required to allow buffer exchange, our extensive preliminary data confirms feasibility. In Aim 1, we will develop a high-pressure chamber compatible with buffer exchange and cryo-vitrification and characterize its performance. In Aim 2, we will develop a method for time-resolved cryo- cooling and validate the system using gold-standard biological samples. Development of HPF-X is an emergent technical opportunity given the advent of nanoscale bioimaging. Importantly, this work goes beyond the current method development regime in cryo-vitrification field because all available HPF devices are commercial. Our custom-built HPF-X in...