Biomedical Studies and Cellular Imaging via Atomic Force Microscopy

我们继续开发我们的生物物理测量系统（生物原子力显微镜（Bio-AFM）平台，石英晶体微天平耗散（QCM-D）和光学显微镜和光谱学），并将这些技术应用于重要的生物医学研究与杰出的NIH校内和校外科学家合作。 在仪器方面，我们已经建立了一个新的高速原子力显微镜从国产和开源组件的扫描速率高达每秒20线。 我们正在继续整合拉曼光谱与生物原子力显微镜，并朝着更广泛的适用性原子力显微镜成像和单分子力光谱（SMFS）的细胞和生物分子样品表征。 在应用方面，我们于本年度的主要合作及显著成果包括： (1)我们一直致力于临床疫苗开发，以增强免疫反应并最终根除疟疾。 我们通过Bio-AFM和相关的生物分析与大卫纳鲁姆博士（疟疾免疫学和疫苗学实验室，NIAID，NIH）和其他合作者研究了更多疟疾候选疫苗和病毒样颗粒载体的大分子结构和纳米机械特性。这些疟疾蛋白抗原和疫苗载体通过重组蛋白生物技术生产，纯化，并以适合人体试验和规模生产的方式表征。使用生物原子力显微镜成像和力光谱学在单个大分子和组装水平的生物物理表征有助于定义这些疫苗结构沿着发展阶段。我们还旨在提高疟疾寄生虫和病原体-宿主相互作用的机制的理解。 (2)我们已经扩大了与Xiaoyuan Chen博士（分子成像和纳米医学实验室，NIBIB），Ashwin Bhirde博士，Peng Huang博士，Dingbin Liu博士和一个国际合作研究团队在多功能纳米医学探针方面的合作。 染料负载的铁蛋白纳米笼，RNA纳米颗粒和碳纳米管/氧化石墨烯为基础的治疗诊断是纳米医学，生物成像和生物标志物检测探针通过生物原子力显微镜研究，并于今年出版。 对于多功能细胞成像和生物医学应用，我们正在研究Bio-AFM和QCM-D方法的更广泛应用，以研究纳米粒子治疗诊断学及其对癌细胞，干细胞和相关生物医学系统的影响。 (3)我们与合作者Ralph Nossal和Dan Sackett博士（NICHD，NIH）以及Eileen Lafer教授（德克萨斯大学健康科学中心，圣安东尼奥）继续进行蛋白质网格蛋白和组装的生物原子力显微镜研究。 网格蛋白是受体介导的内吞作用和细胞内运输的关键蛋白。 今年，进一步的Bio-AFM和QCM-D测量已经被用于表征网格蛋白及其组装结构，以及与对细胞功能重要的几种伴侣蛋白的相互作用。我们与Ling-gang Wu博士（NINDS）及其同事发表了关于致密核心囊泡胞吐和胞吞作用中的膜结构变化及其对大脑突触传递的影响。 (4)我们与NIDCR科学家Andrew Doyle博士和Kenneth Yamada（细胞和发育生物学实验室，NIDCR）合作，通过Bio-AFM力谱研究组织特异性细胞外基质和重组基质样凝胶的纳米力学和结构特性。与Raimon Sunyer博士（NICHD，目前在西班牙加泰罗尼亚生物工程研究所）合作，我们正在定义最佳的力谱方法来探索组织模拟凝胶基质及其与细胞粘附和迁移的相关性。 (5)在今年的其他持续合作中，我们与Yawen Bai博士（分子细胞生物学实验室，NCI）及其同事研究了蛋白质和DNA相互作用以及核小体结构和动力学，这在基因调控和疾病机制中至关重要。 我们与乔治华盛顿大学的邱祥云教授及其团队合作进行DNA交联及相关研究。 与其他合作者（例如NHLBI的Richard Hendler博士）一起，我们已经推进了我们的生物原子力显微镜和几种蛋白质纤维组装的生物物理研究，例如与阿尔茨海默病有关的淀粉样β纤维。 (6)今年，我们启动了新的合作项目。我们已经开始与Qi Lu、Hacene Boukari和Yuriy Markushin博士合作开展INBRE试点项目，研究纳米颗粒对脂质结构域组织的影响，以预防和治疗癌症。我们已经开始与Yun-Xing Wang博士（NCI）及其同事合作，确定关键人类免疫缺陷病毒（HIV）RNA及其与辅因子的复合物的高分辨率结构，并开发治疗方法。

We continue to develop our biophysical measurement systems (biological atomic force microscopy (Bio-AFM) platforms, Quartz Crystal Microbalance-Dissipation (QCM-D), and optical microscopy and spectroscopy), and to apply these technologies to important biomedical investigations in collaboration with outstanding NIH intramural and extramural scientists. On the instrumentation front, we have built a new high-speed AFM from home-made and open-source components with scan rates up to twenty lines per second. We are continuing an integration of Raman spectroscopy with Bio-AFM, and working toward broader applicability of AFM imaging and single molecule force spectroscopy (SMFS) for cellular and bio-molecular sample characterizations. On the application front, our major collaborations and notable results in this year include: (1) We have maintained our commitment to work on clinical vaccine development toward enhanced immunological response and eventual eradication of malaria. We have investigated the macromolecular structure and nanomechanical properties of more malaria vaccine candidates and virus-like-particle carriers via Bio-AFM and related bioanalysis with Dr. David Narum (Laboratory of Malaria Immunology and Vaccinology, NIAID, NIH), and other collaborators. These malaria protein antigens and vaccine carriers are produced via recombinant-protein biotechnology, purified, and characterized in a manner suitable for human trials and scale-up production. Biophysical characterization at single macromolecule and assembly level using Bio-AFM imaging and force spectroscopy are helping define these vaccine constructs along the developmental phases. We also aim to improve mechanistic understanding of the malaria parasites and pathogen-host interactions. (2) We have expanded our collaboration on multifunctional nanomedicine probes with Dr. Xiaoyuan Chen (laboratory of Molecular Imaging and Nanomedicine, NIBIB), Dr. Ashwin Bhirde, Dr. Peng Huang Dr. Dingbin Liu, and an international team of co-investigators. Dye-loaded ferritin nanocages, RNA-nanoparticle and carbon nanotube/graphene oxide based theranostics are among nanomedicine, Bioimaging and biomarker detection probes studied via Bio-AFM and published this year. Toward multifunctional cellular imaging and biomedical applications, we are examining broader applications of Bio-AFM and QCM-D methodology for investigating nanoparticle theranostics and their impact on cancer cells, stem cells, and related biomedical systems. (3) We have continued our Bio-AFM studies of protein clathrin and assemblies with collaborators including Drs. Ralph Nossal and Dan Sackett(NICHD, NIH), and Prof. Eileen Lafer (Univ. Texas Health Sciences Center, San Antonio). Clathrin is a key protein for receptor-mediated endocytosis and intracellular trafficking. Further Bio-AFM and QCM-D measurements have been pursued this year to characterize clathrin and its assembled structures, as well as interaction with several partner proteins important to the function of cells. We published with Dr. Ling-gang Wu (NINDS) and coworkers on membrane structural changes in exocytosis and endocytosis of dense-core vesicles and their implications for synaptic transmission in brain. (4) We have collaborated with NIDCR scientists, Drs. Andrew Doyle and Kenneth Yamada (Laboratory of Cell and Developmental Biology, NIDCR), on nanomechanics and structural properties of tissue specific extracellular matrices and reconstituted matrix-like gels via Bio-AFM force spectroscopy. Collaborating also with Dr. Raimon Sunyer (NICHD and currently at Institute for Bioengineering of Catalonia, Spain), we are defining the best force spectroscopy approaches to explore tissue-mimicking gel matrices and their relevance to cell adhesion and migration. (5) Among other continuing collaborations this year, we have investigated further protein and DNA interactionnns and nucleosome structure and dynamics, critically important in gene regulation and disease mechanisms, with Dr. Yawen Bai (Laboratory of Molecular Cell Biology, NCI) and coworkers. We have collaborated with Prof. Xiangyun Qiu (George Washington University) and his group on DNA cross-linking and related studies. And with other groups of collaborators (e.g. Dr. Richard Hendler, NHLBI), we have advanced our Bio-AFM and biophysical studies of several protein fiber assemblies, such as amyloid-beta fibers implicated in the Alzheimer's disease. (6) We have initiated new collaborative projects this year. We have started to collaborate on an INBRE pilot project with Drs. Qi Lu, Hacene Boukari and Yuriy Markushin on the effects of nanoparticles on the organization of lipid domains toward cancer prevention and treatment. We have started to work with Dr. Yun-Xing Wang (NCI) and colleagues on determining high-resolution structures of a key human immunodeficiency virus(HIV) RNA and its complex with co-factors and developing therapeutics.