Atomic Force Microscopy Instrumentation and Applications

原子力显微镜(AFM)在研究生物分子和组织的超微结构和功能特性方面正变得越来越有用。DBEPS仪器研究和开发资源正在加强NIH的AFM能力，以支持IC科学项目的不同需求。已经获得了一套原子力显微镜仪器系统，并正在对其进行改造，以便对生物样品进行测量。已经开发出一种灌注室，使单个细胞能够被保留以进行检查，并允许操纵细胞外环境。还开发了一种温度控制的环境室，以允许对表现出温度相关性质的样品进行检查。该燃烧室能够在10至40摄氏度的温度范围内保持0.1度的稳定性。利用DBEPS在光学、电子学、机械设计和其他领域的专业知识，人们正在寻求相关的仪器和定量分析方法，以促进AFM技术的发展，并将其应用于解决新的生物医学问题。协作性的壁内生物学项目包括对亚细胞蛋白质运输(NICHD)和表面修饰蛋白质相互作用动力学(NIDDK)至关重要的蛋白质网状蛋白及其组装体的粘弹性能量学的研究。原子力显微镜与近场扫描光学显微镜(NIAID)的集成正在通过与NIST的合作协议而进行，以开发和调整现有的仪器以应用于生物学问题。我们新方法的初步实施已经成功地应用于用于校准和验证的标准样品。新的AFM组件正在被获取和集成，以扩展我们的设施能力，以满足来自NIH的内部研究人员的不同需求。 这项技术正被应用于研究与囊泡形成和囊泡运输相关的机械特性。在这个特定的项目中，我们研究了网状蛋白三离子在直径约100纳米的多面体涂层中的组装，这被认为在受体介导的内吞作用和跨高尔基体网络的细胞内运输中发挥了核心作用。为了充分了解该涂层在囊泡形成的动态控制中的作用，需要了解该涂层的机械性能。这里的目的是测量生物流体环境中包被和未包覆的囊泡的机械性能。我们已经开发了一种方案，利用50-100 pN范围的变形力，可以用AFM定量地探索这两种囊泡的纳米力学。通过更好的仪器参数校准和囊泡样品制备，测量正在得到改进。

The atomic force microscope (AFM) is becoming increasingly useful for studying ultra-structure and functional properties of biological molecules and tissue. The DBEPS Instrumentation Research and Development Resource is strengthening the AFM capabilities at NIH to support the diverse needs of IC scientific projects. An AFM instrument system has been obtained and is being adapted to allow measurements to be made of biological samples. A perfusion chamber has been developed to enable a single cell to be retained for examination and permitting manipulation of the extra-cellular environment. 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 Celsius. Using DBEPS expertise in optics, electronics, mechanical design, and other areas, associated instrumentation and quantitative analysis methods are being pursued to advance AFM technology and apply it to solve novel biomedical problems. 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 (NICHD) and surface modified protein interaction dynamics (NIDDK). Integration of AFM with near-field scanning optical microscopy (NIAID) is being pursued through a collaborative agreement with NIST to develop and adapt existing instrumentation for application to biological problems. Preliminary implementation of our novel methodology has been successful when applied to standard samples for calibration and validation. New AFM components are being acquired and integrated to expand our facility capabilities towards meeting the varying needs of intramural researchers from across NIH. This technology is being applied to studying 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 which is 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. The objective here is to measure the mechanical properties of clathrin-coated and uncoated vesicles in biological fluid environments. We have developed a scheme by which the nanomechanics of both vesicle types can be quantitatively explored by AFM, employing a deformation force in the 50-100 pN range. The measurements are being refined via better instrument parameter calibrations and vesicle sample preparations.