An Improved Atomic Force Microscope for Biomedical Applications: Deep AFM for lar

描述（由申请人提供）：需要一种新的工具来探索有助于组织力学性能的纳米级机制。这些纳米级机制对于理解骨脆性、椎间盘退变、骨关节炎和其他疾病至关重要。原子力显微镜（AFM）具有成像和操纵纳米结构的能力，已成为该领域的有力工具。但它对研究组织样本有一个主要限制：大多数组织样本太粗糙，无法用AFM悬臂进行成像。在这里，我们建议超越悬臂梁，采用一种新颖的深层AFM探针，该探针可以垂直深入组织样品的地形。提出的研究目标是建立新一代原子力显微镜（Deep afm）的第一个原型，这将使afm的成像范围至少增加一个数量级，然后使用这些Deep afm来探索组织结构和纳米力学，目的是在足够详细的水平上了解组织变性所涉及的分子和纳米级过程，从而为新疗法的开发提供信息。由于许多潜在的应用，所提出的工作的总体影响和相关性是广泛的，然而，由于我们目前在骨诊断方面的进展和工作，我们将主要关注骨的纳米级断裂力学。我们提出了三个相关的目标，包括这类新型原子力显微镜的开发和表征，以及它们在临床相关骨组织样本中的应用。具体目标1是开发用于大规模扫描和纳米力学的深度AFM I。它将实现骨在缓冲液中裂纹扩展的成像，以及空间分辨力谱、纳米操作和压痕来测量局部纳米力学性能。具体目标2是开发用于高分辨率，大规模扫描和纳米力学的Deep AFM II。高分辨率的Deep AFM II将能够成像纳米级骨折裂纹的起源，其分辨率可与扫描电子显微镜相媲美，但无需从缓冲液中取出样品。因此，它将有可能成像纳米级的过程，发生在中间阶段的裂纹扩展过程。它也将有可能执行空间分辨力光谱，纳米操作和压痕来测量局部纳米力学性能。具体目标3是使用深度AFM来推进我们对骨折的纳米级机制和降低骨折风险的方法的理解。有了深度AFM，我们可以通过了解抵抗骨折的分子和纳米级机制，继续朝着临床降低骨折风险的长期目标迈进。

DESCRIPTION (provided by applicant): A new tool is necessary to explore the nanoscale mechanisms that contribute to tissue mechanical properties. These nanoscale mechanisms are of key importance in understanding bone fragility, intervertebral disc degeneration, osteoarthritis, and other diseases. The Atomic Force Microscope, AFM, with its ability to both image and manipulate nanostructures, has been a powerful tool in this area. But it has a major limitation for studying tissue samples: most tissue samples are too rough to be imaged with AFM cantilevers. Here we propose to move beyond cantilevers to a novel Deep AFM probe that enables a vertical approach deep into the topography of tissue samples. The objectives of the proposed research are to build the first prototypes of a new generation of Atomic Force Microscopes, Deep AFMs, that will increase imaging range of AFMs by at least one order of magnitude, and then to use these Deep AFMs to explore tissue structures and nanomechanics with a goal of understanding the molecules and nanoscale processes involved in tissue degeneration at a sufficient level of detail to inform development of new therapies. The overall impact and relevance of the proposed work is broad due to the numerous potential applications, however, because of our current progress and work on bone diagnostics, we will focus primarily on the nanoscale fracture mechanics of bone. We propose three related aims that include both the development and characterization of this new class of AFMs as well as their application in clinically relevant bone tissue samples. Specific Aim 1 is to develop Deep AFM I for very large scale scanning and nanomechanics. It will enable imaging of crack propagation in bone submerged in buffer as well as spatially resolved force spectroscopy, nanomanipulation and indentation to measure local nanomechanical properties. Specific Aim 2 is to develop Deep AFM II for high resolution, large scale scanning and nanomechanics. The higher resolution of Deep AFM II will enable imaging the nanoscale origin of bone fracture cracks with resolution comparable to Scanning Electron Microscopy, but without ever removing the sample from buffer. Thus it will be possible to image nanoscale processes that occur at intermediate stages of the crack growth process. It will also be possible to perform spatially resolved force spectroscopy, nanomanipulation and indentation to measure local nanomechanical properties. Specific Aim 3 is to use Deep AFM to move forward in our understanding of the nanoscale mechanisms of bone fracture and ways to reduce bone fracture risk. With Deep AFM, we can continue to move toward a long term goal of clinically decreasing the component of bone fracture risk by understanding the molecules and nanoscale mechanisms that resist bone fracture.