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

六维单分子纳米显微镜用于阐明生物分子的动态组织

• 批准号: 10623390
• 负责人: Matthew D Lew
• 金额: $ 46.62万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-08-15 至 2028-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10623390
• 关键词: 3-Dimensional; Algorithm Design; Alzheimer' s Disease; Amyloid; Amyloidosis; Amyotrophic Lateral Sclerosis; Architecture; Binding; Biocompatible Materials; Biophysical Process; Cells; Complex; Data; Development; Diagnostic; Dimensions; Evolution; Fluorescence; Funding; Gene Expression Regulation; Goals; Heterogeneity; Image; In Vitro; Individual; Lighting; Maps; Measurement; Measures; Methods; Microscope; Modeling; Molecular; Motion; Nanoscopy; Nucleic Acids; Peptides; Phase; Photons; Physical condensation; Positioning Attribute; Process; Proteins; Pupil; Research; Resolution; Scientist; Speed; Structure; Techniques; Technology; Therapeutic; Time; Visualization; cytotoxicity; deep learning; driving force; fluorophore; imaging capabilities; imaging system; insight; intercellular communication; learning strategy; molecular dynamics; molecular imaging; nanoscale; network architecture; new technology; novel; programs; protein aminoacid sequence; quantum; recruit; self assembly; single molecule; spatiotemporal; tool

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.

7.项目摘要 拟议的MIRA资助的研究组合的目标是发现 纳米级的单个生物分子影响它们的集体功能和复杂的组织， 生物物理过程拟议的研究计划将继续开发6D单- 分子（SM）成像（3D位置和3D方向）与组织的机制研究 纳米级的生物分子相互作用。重要的是，拟议的科学目标协同刺激了 发展有影响力的成像能力，这些新能力将反过来克服障碍， 新的重要的科学轨迹要追求。将开展四个广泛的研究重点。 推力1将开发智能自适应6D纳米显微镜。先前的研究表明，固定成像系统 不能以最好的量子极限精度测量所有可能的分子旋转运动。因此，本发明的目的是， 将集成动态照明和荧光调制硬件，以使成像系统能够 随着数据的收集而调整。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， 生产智能显微镜，使测量是不可能的，即使与当前的最先进的 纳米显微镜推力2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。这些不均匀性控制着重要的过程，如相分离， 但是当前的技术具有足够的时空分辨率来解析机械细节。时变 照明，单光子计数和直接瞳孔成像将被集成，以可视化这些动态，使用 比现有技术方法少10倍的发射光子，因此速度快10倍。 推力3将利用6D纳米显微镜的发展来阐明自组装的动态分子结构。 组装肽和天然淀粉样蛋白。关键的是，科学家们必须解开 肽序列、二级结构、组装结构和聚集条件，以产生新的 用于诊断和治疗的生物材料，以及阐明淀粉样蛋白中细胞毒性的机制 疾病瞬时结合荧光团的6D位置和取向将可视化动态的荧光团的位置和取向。 单个肽组装体在体外以及与纳米级活细胞相互作用时的组织 分辨率Thrust 4将利用6D SM跟踪的发展来可视化异构网络 生物分子凝聚物中的结构，系综测量无法检测到。6D位置和 荧光探针的方向将用于表征粘着剂和间隔物的网络结构 在冷凝物内，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如何在整个相分离过程中被募集和重组， 导致对生物分子凝聚物的形成和时空演化的机械见解。