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. PUBLIC HEALTH RELEVANCE: Bone fragility, intervertebral disc degeneration, cancer, atherosclerosis, osteoarthritis, and tooth decay all involve changes in tissue mechanical properties at the nanoscale. The proposed Deep Atomic Force Microscope, Deep AFM, is designed to investigate these changes with the goal of understanding how to prevent and even reverse undesirable changes for tissues in general and bone in particular. This work will be synergistic with our ongoing collaboration with physicians on diagnosing bone fragility due to bone tissue degeneration in patients.