Six-Dimensional Single-Molecule Nanoscopy for Elucidating the Dynamic Organization of Biomolecules

7.项目总结 提出的米拉资助的研究项目组合的目标是发现 纳米级的单个生物分子影响它们在复合体中的集体功能和组织 生物物理过程。所提出的研究计划整合了6D单通道的持续开发 分子(SM)成像(3D位置和3D方向)与组织的机制研究 纳米尺度上的生物分子相互作用。重要的是，提出的科学目标协同作用地刺激了 开发有影响力的成像功能，这些新功能将反过来克服障碍，实现 有待探索的新的重大科学轨迹。将开展四项广泛的研究工作。 推力1号将开发智能自适应6D纳米显微镜。以前的研究表明，固定的成像系统 无法以最好的量子限制精度测量所有可能的分子旋转运动。因此， 动态照明和荧光调制硬件将集成在一起，使成像系统能够 随着数据的收集而进行调整。将模型驱动的设计算法与数据驱动的深度学习方法相融合将 生产智能显微镜，实现即使在目前最先进的情况下也不可能进行的测量 纳米显微镜。推力2号将开发高速6D SM跟踪，以绘制分子中的空间异质性 生物分子之间的相互作用。这些异质性支配着重要的过程，如相分离， 但目前的技术有足够的时空分辨率来解决机械细节问题。时变 照明、单光子计数和直接瞳孔成像将被集成在一起，以使用 发射光子减少10倍，因此速度比最先进的方法快10倍。 推进器3将利用6D纳米技术的发展来阐明自 组装多肽和天然淀粉样蛋白。至关重要的是，科学家们必须理清 肽序列、二级结构、组装结构和聚集条件，以创建新的 用于诊断和治疗的生物材料，以及阐明淀粉样蛋白的细胞毒性机制 疾病。瞬时结合的荧光团的6D位置和取向将可视化动态 单个多肽组件在体外以及与纳米级活细胞相互作用时的组织 决议。推力4将利用6D SM跟踪的发展来可视化异类网络 生物分子凝聚体中的整体测量未能检测到体系结构。6D位置和 荧光探针的取向将被用来表征粘合剂和间隔物的网络结构 在凝析油中，从而可视化相分离的驱动力。六维SM纳米显微镜 还将直接观察蛋白质在相分离过程中是如何招募和重组的， 导致了对生物分子凝聚体的形成和时空演化的机械洞察。

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 the phase separation process, leading to mechanistic insights into the formation and spatiotemporal evolution of biomolecular condensates.