Atomic Force Microscopy Instrumentation and Applications

一个新的AFM单分子力谱（SMFS）仪器（ForceRobot）已被集成到我们的核心设施。其他原子力显微镜组件正在收购和整合，以扩大我们的设施的能力，以满足来自全国卫生研究院的内部研究人员的不同需求。合作的校内生物学项目包括研究蛋白质网格蛋白及其组装体的粘弹性能，这对亚细胞蛋白质运输很重要（与NICHD合作），大肠杆菌衍生恶性疟原虫裂殖子表面蛋白3和环子孢子蛋白（CSP）作为潜在的人类疟疾疫苗成分的结构研究（与NIAID），视紫红质在天然盘膜表面的组织（与NIAAA），表面修饰蛋白质相互作用动力学（与NIDDK），基因调控途径中的DNA-蛋白质相互作用（用NCI），细胞色素c吸附到阴离子支持的双层（用NIDDK），和测量的哺乳动物耳蜗中的盖膜的机械性质（用NIDCD）。 我们AFM技术开发的一个重点是将光谱学与AFM结合起来，用于多模态超灵敏纳米样品表征。特别是，全内反射荧光显微镜（TIRFM）和共焦拉曼显微镜与合适的AFM仪器相结合，观察和关联多个结构特性。灵敏的检测设备、配置和振动隔离设置正被并入以实现单个生物分子的分辨率和灵敏度。我们正在通过研究量子点（q-dots）和碳纳米管来优化我们的仪器，量子点是具有革命性荧光性能的纳米晶体，碳纳米管在纳米技术和相关领域都具有巨大的潜力。当量子点与环境相互作用时，我们的TIRFM-AFM和Raman-AFM被用来观察它们的微内核和生物相容性涂层。我们的目标是通过将高分辨率拓扑，机械和静电配置文件与光学特性（如荧光光谱，闪烁性（即闪烁）和光稳定性）相关联来了解单粒子行为。 我们已经扩大了我们的理解，碳纳米管使用同时AFM成像和尖端增强拉曼光谱（TERS）映射。 增强AFM能力的技术开发的其他示例包括使用弹性体微通道在AFM衬底上临时创建微器件。因为在这样的微通道中的流动是层流，所以所有的混合都是扩散的，这允许我们在几百微米的距离上创建良好控制的缓冲液梯度，无论是离子强度还是pH。这允许快速采样的缓冲液条件的样品沉积在AFM基板上，并提供了大幅减少所需的时间，以优化AFM成像的吸附条件的潜力。以前，还开发了温度控制的环境室，以允许对表现出温度依赖性的样品进行检查。该腔室能够在10至40摄氏度的温度范围内保持0.1度的稳定性。已经开发了灌注室，以使单个细胞能够被保留用于检查并允许操纵细胞外环境。 我们的AFM应用的一个例子是与囊泡形成和囊泡运输的机械性能的研究。在这个特定的项目中，我们研究组装成多面体外套的网格蛋白triskelions约100纳米的直径，被认为是发挥核心作用，受体介导的内吞作用和细胞内运输的trans-Golgi网络。为了充分理解囊泡形成的动态控制中的外套的功能，需要网格蛋白外套的机械性能的知识。为了解开他们的分子结构的复杂性，我们研究网格蛋白包被的囊泡（CCV）的机械性能，同时开发新的原子力显微镜（AFM）和相关的分析方案。最近的进展包括在流体缓冲液下直接AFM可视化三聚氰胺离子分子的灵活性。 我们的新SMFS揭示，第一次，一系列的内部充满活力的障碍，其特征三氟甲磺酸离子重链折叠和展开，以及相关的蛋白质序列的7个重复的145氨基酸基序和众多的30 aa发夹。这些结构域的动态稳定性进行了研究。

A new AFM single molecule force spectroscopy (SMFS) instrument (ForceRobot) has been integrated into our core facility. Other AFM components are being acquired and integrated to expand our facility's capabilities of meeting the varying needs of intramural researchers from across NIH. Collaborative intramural biological projects include the investigation of the viscoelastic energetics of the protein clathrin and its assemblies that are important to subcellular protein trafficking (in collaboration with NICHD), structure studies of an Escherichia coli derived Plasmodium falciparum Merozoite Surface Protein 3 and circumsporozoite protein (CSP) as potential human malaria vaccine components (with NIAID), organization of rhodopsin on the surface of native disk membranes (with NIAAA), surface modified protein interaction dynamics (with NIDDK), DNA-protein interactions in gene regulation pathways (with NCI), cytochrome c adsorption to anionic supported bilayers (with NIDDK), and measured mechanical properties of the tectorial membrane in the mammalian cochlea (with NIDCD). A strong focus of our AFM technology development is combining optical spectroscopies with AFM for multimodal ultra-sensitive nanometric sample characterizations. In particular, total internal reflection fluorescent microscopy (TIRFM) and confocal Raman microscopy are combined with suitable AFM instruments to observe and correlate multiple structural properties. Sensitive detection devices, configurations, and vibration-isolation setups are being incorporated toward achieving single biomolecule resolution and sensitivity. We are optimizing our instrumentations through studies of quantum dots (q-dots), which are nanocrystals with revolutionary fluorescence performance, and carbon nanotubes, both with huge potential in nanotechnology and related fields. As q-dots interact with their environment, our TIRFM-AFM and Raman-AFM are used to observe their nanocrystal cores and biocompatible coatings. We aim to understand the single particle behavior by correlating high resolution topological, mechanical, and electrostatic profiles with optical properties, such as fluorescent spectra, intermittency (i.e. blinking), and photostability. We have expanded our understanding of carbon nanotubes using simultaneous AFM imaging and tip enhanced Raman spectroscopy (TERS) mapping. Other examples of technology development to enhance AFM capabilities include the use of elastomeric microchannels to temporarily create a microdevice on an AFM substrate. Because flow in such microchannels is laminar, all mixing is diffusive, which allows us to create a well-controlled buffer gradient, either in ionic strength or pH, over a distance of a few hundred microns. This permits rapid sampling of buffer conditions for sample deposition on AFM substrates and offers the potential of reducing substantially the time required to optimize adsorption conditions for AFM imaging. Previously, a temperature-controlled environmental chamber has also been developed to allow samples that exhibit temperature dependent properties to be examined. The chamber is capable of 0.1 degree stability within a temperature range from 10 to 40 degrees Celsius. A perfusion chamber has been developed to enable a single cell to be retained for examination and permitting manipulation of the extra-cellular environment. One example of our AFM applications is to the study of the mechanical properties associated with vesicle formation and vesicular trafficking. In this particular project we examine the assembly of clathrin triskelions into polyhedral coats of about 100-nanometer diameter that are believed to play a central role in receptor-mediated endocytosis and intracellular trafficking from the trans-Golgi network. Knowledge of the mechanical properties of the clathrin coat is needed in order to fully understand the function of the coat in the dynamical control of vesicle formation. To unravel the intricacy of their molecular constructs, we examine the mechanical properties of clathrin-coated vesciles (CCVs), while developing new schemes of atomic force microscopy (AFM) and related analyses. More recent advancement includes direct AFM visualizations of triskelion molecular flexibility under fluid buffers. Our new SMFS reveals, for the first time, a series of internal energetic barriers that characterize triskelion heavy chain folding and unfolding, well correlated with the protein sequence of both the seven repeating 145aa motifs and numerous 30aa hairpins. The dynamic stability of these structural domains has been examined.