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与NIH内外科学家合作的一些生物医学和生物学研究。 在仪器方面，我们正在继续将拉曼光谱与生物原子力显微镜相结合，并致力于将原子力显微镜成像和单分子力光谱（SMFS）用于细胞和生物医学样品表征。 在应用方面，我们于本年度的主要合作及显著成果包括： (A)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正在通过重组蛋白生物技术生产、纯化并以适合人体试验和大规模生产的方式进行表征。使用AFM成像和力谱在单个大分子水平上的生物物理表征有助于通过其发展阶段来定义这些生物制剂。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。 (B)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，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。 基于氧化石墨烯的治疗诊断学和基于金纳米颗粒的成像和生物标志物检测探针的新结果是今年发表的。 基于碳纳米管、纳米点和其他纳米结构的治疗诊断学的其他结果已经进一步发展到多细胞成像和生物医学应用。我们正在研究Bio-AFM和QCM-D方法在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方面的更广泛应用。 (C)我们与合作者Ralph Nossal博士（NICHD，NIH）和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质，我们在今年使用单分子力谱（SMFS）报道了网格蛋白-网格蛋白相互作用和网格蛋白结构域内折叠能量学。 进一步的AFM和QCM-D测量被追求来表征网格蛋白及其组装结构，以了解其从酵母到人类的功能。 (D)我们与NIDCR科学家Andrew Doyle博士，Jill Harunaga女士和Kenneth Yamada博士（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。我们还与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，定义了最佳的力谱方法，以探索生物基质及其在细胞生物学和组织分化中的意义。 (E)在今年的其他合作中，我们与Yawen Bai博士（分子细胞生物学实验室，NCI）及其同事研究了核小体结构和动力学中涉及的蛋白质和DNA相互作用，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邱祥云教授及其团队合作进行DNA交联及相关研究。 我们还与Richard Hendler博士（NHLBI），Katelyn Nagy女士（NCI）和Joel Schneider博士（NCI）等合作者一起推进了我们对不同蛋白质纤维组装体的Bio-AFM和生物物理研究。

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 a number of biomedical and biological investigations in collaboration with NIH intramural and extramural scientists. On the instrumentation front, we are continuing an integration of Raman spectroscopy with Bio-AFM, and working toward higher speed and broader applicability of AFM imaging and single molecule force spectroscopy (SMFS) for cellular and biomedical sample characterizations. On the application front, our major collaborations and notable results in this year include: (A) 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 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 being produced via recombinant-protein biotechnology, purified, and characterized in a manner suitable for human trials and scale-up production. Biophysical characterization at single macromolecular level using AFM imaging and force spectroscopy are helping define these biologics through their developmental phases. We also aim to improve mechanistic understanding of the malaria parasites and pathogen-host interactions. (B) We have expanded our collaboration on multifunctional nanomedicine probes with Dr. Xiaoyuan Chen (laboratory of Molecular Imaging and Nanomedicine, NIBIB), Dr. Ashwin Bhirde, Dr. Dingbin Liu, and an international team of co-investigators. New results on graphene oxide based theranostics and gold nanoparticle based imaging and biomarker detection probes are among those published this year. Other results on theranostics based on carbon nanotubes, nanodots and other nano-constructs have been developed further toward multiple 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. (C) We have continued our Bio-AFM studies of protein clathrin and assemblies with collaborators including Dr. Ralph Nossal (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 and we reported on clathrin-clathrin interactions and intra-clathrin domain folding energetics using single molecule force spectroscopy (SMFS) during this year. Further AFM and QCM-D measurements are been pursued to characterize clathrin and its assembled structures toward understanding its function from yeast to human. (D) We have collaborated with NIDCR scientists, Dr. Andrew Doyle, Ms. Jill Harunaga and Dr. 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 biological matrices and their implications in cell biology and tissue differentiation. (E) Among other collaborations this year, we have investigated protein and DNA interactions involved in 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 investigations. We have also advanced our Bio-AFM and biophysical studies of different protein fiber assemblies with collaborators including Dr. Richard Hendler(NHLBI), Ms. Katelyn Nagy (NCI), and Dr. Joel Schneider (NCI).