Feasibility: Non-Contact Cryo-Atomic Force Microscope High Resolution Bioimaging

描述（由申请人提供）：纳米科学和纳米技术倡议的主要目标之一是开发能够“有效检测和分析与生物医学相关的纳米级实体”的通用方法。目前，生物膜中的超分子复合物、具有内在柔性和/或异质性的结构和整合膜蛋白仍然是一个重大的技术挑战。因此，提取“定量信息”的任务（例如，从生物纳米级材料和机器“中提取结构细节）需要开发比那些必须依靠系综平均来实现足够高的分辨率的方法更合适的方法。在这里，我们建议开发一种在低温下的原子力显微镜（AFM），以在单个复合物和纳米级生物机器上实现1 nm或更好的空间分辨率。其基本思想是将非接触式成像的高灵敏度与天然结构的冷冻保存相结合。只有采用这种方法，AFM针尖的锐度才能在成像过程中免受退化，这是超高分辨率成像的一个最关键因素。该R21应用程序的直接目标是通过实验确定这种方法的可行性，并测量鲁棒仪器设计所需的基本参数。如果我们成功地将这一概念发展成一种实用的仪器，这种仪器将是一种能够分辨微小表面结构细节的强大方法（例如，二级结构）的单个复合物，不能超越任何其他目前可用的方法。

DESCRIPTION (provided by applicant): One of the major objectives of the nanoscience and nanotechnology initiative is to develop general methodologies that can "effectively detect and analyze nanoscale entities of relevance to biomedicine". At present, supra-molecular complexes, structures with intrinsic flexibilities and/or heterogeneities and integral membrane proteins in the biological membrane remain a significant technological challenge. As such, the task of extracting "quantitative information (e.g., structural details) from biological nanoscale materials and machines" requires the development of more appropriate methods than those that must rely on ensemble averaging to achieve a high enough resolution. Here, we propose to develop an atomic force microscope (AFM) at cryogenic .temperatures to achieve a spatial resolution of 1 nm or better on individual complexes and nanoscale biological machines. The essential idea is to combine the high sensitivity of non-contact imaging and cryo-preservation of the native structure. Only with such an approach can the sharpness of an AFM tip be protected from degradation during imaging, a factor most critical for ultra-high resolution imaging. The immediate goal of this R21 application is to experimentally determine the feasibility of this approach and measure the fundamental parameters necessary for the design of robust instrumentation. If we succeed in developing this concept into a practical apparatus, such instrumentation will be a powerful method capable of resolving minute surface structural details (e.g., secondary structures) of individual complexes that cannot be surpassed by any other currently available approach.