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“有效地检测和分析与生物医学相关的纳米级实体”的一般方法。目前，生物膜中的超分子复合体、具有内在柔性和/或异质性的结构以及完整的膜蛋白仍然是一个重大的技术挑战。因此，从生物纳米材料和机器中提取“定量信息(例如，结构细节)”的任务需要开发更合适的方法，而不是那些必须依赖集合平均才能达到足够高分辨率的方法。在这里，我们建议开发一种在低温下的原子力显微镜(AFM)，以便在单个复合体和纳米级生物机器上达到1 nm或更高的空间分辨率。其基本思想是将非接触成像的高灵敏度与自然结构的冷冻保存结合起来。只有使用这种方法，AFM针尖的锐度才能在成像过程中避免退化，这是超高分辨率成像最关键的因素。此R21应用程序的直接目标是通过实验确定该方法的可行性，并测量设计健壮仪器所需的基本参数。如果我们成功地将这一概念发展成实用的仪器，这种仪器将是一种强大的方法，能够解决单个络合物的微小表面结构细节(例如，二级结构)，这是目前任何其他可用的方法都无法超越的。

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.