7. PROJECT SUMMARY The goal of the proposed MIRA-funded research portfolio is to discover how dynamic interactions between individual biomolecules at the nanoscale influence their collective function and organization in complex biophysical processes. The proposed research program integrates continued development of 6D single- molecule (SM) imaging (3D positions and 3D orientations) with mechanistic studies of the organization of biomolecular interactions at the nanoscale. Importantly, the proposed scientific goals synergistically spur the development of impactful imaging capabilities, and these new capabilities will in-turn overcome barriers to enable novel significant scientific trajectories to be pursued. Four broad research thrusts will be pursued. Thrust 1 will develop smart adaptive 6D nanoscopy. Previous studies have shown that fixed imaging systems cannot measure all possible molecular rotational motions with the best-possible quantum-limited precision. Thus, dynamic illumination and fluorescence modulation hardware will be integrated to enable the imaging system to adapt as data is collected. Fusing model-driven design algorithms with data-driven deep learning methods will yield smart microscopes that enable measurements that are not possible even with current state-of-the-art nanoscopes. Thrust 2 will develop high-speed 6D SM tracking to map spatial heterogeneities in molecular interactions between biomolecules. These heterogeneities govern important processes like phase separation, but current techniques have sufficient spatiotemporal resolution to resolve mechanistic details. Time-varying illumination, single-photon counting, and direct pupil imaging will be integrated to visualize these dynamics using 10x fewer emission photons and thus 10x faster speed than state-of-the art methods. Thrust 3 will leverage developments in 6D nanoscopy to elucidate dynamic molecular architectures of self- assembling peptides and natural amyloidogenic proteins. Critically, scientists must disentangle the effects of peptide sequence, secondary structure, assembly architecture, and aggregation conditions to create new biomaterials for diagnostics and therapeutics, as well as to elucidate the mechanisms of cytotoxicity in amyloid diseases. The 6D positions and orientations of transiently binding fluorophores will visualize the dynamic organization of individual peptide assemblies both in vitro and as they interact with living cells with nanoscale resolution. Thrust 4 will leverage developments in 6D SM tracking to visualize heterogeneous network architectures within biomolecular condensates that ensemble measurements fail to detect. The 6D positions and orientations of fluorogenic probes will be used to characterize the network architecture of stickers and spacers within the condensate, thereby visualizing the driving forces of phase separation. Six-dimensional SM nanoscopy will also directly observe how proteins are recruited and reorganized throughout th...