Atomic force microscope (AFM) has emerged as a powerful tool in the last two decades to study biological materials at the nanoscale. The high resolution imaging capability and high precision force sensitivity of AFM makes it a unique tool for the assessment of characteristic properties of extremely small and soft biological materials.In this dissertation, AFM was utilized to systematically study individual collagen fibrils and cortical bone samples at the nanoscale with the focus on their mechanical and electromechanical properties. Furthermore, a new nanoneedle-based AFM technique was demonstrated to image soft materials such as membrane of living cells in physiological conditions. As the most abundant protein in mammals, type I collagen is a fibrous protein (with in diameter) functioning as one of the main components of bone, tendon, skin, and cornea. In the structural organization of collagen molecules within a collagen fibril there exist alternating zones of gap and overlap in the axial direction of a collagen fibril with a periodicity. This special microstructure has been shown to be of significant importance in multi-functionality of collagen fibrils in tissues with different mechanical requirements. In this dissertation, using high resolution nanoindentation with AFM, nanomechanical heterogeneity along the axial direction of a collagen fibril was revealed; it was shown that the gap and overlap regions have significantly different elastic and energy dissipation properties, correlating the significantly different molecular structures in these two regions. It was further shown that such subfibrillar heterogeneity holds in collagen fibrils inside bone and might be intrinsically related to the excellent energy dissipation performance of bone. Using piezoresposne force microscopy (PFM), the electromechanical properties of a collagen fibril was probed and it was revealed that a single collagen fibril behaves predominantly as a shear piezoelectric material, and has unipolar axial polarization throughout its entire length. Furthermore, it was revealed that there existed an intrinsic piezoelectric heterogeneity within a collagen fibril coinciding with the periodic variation of its gap and overlap regions. This piezoelectric heterogeneity persisted for the collagen fibrils embedded in bone, bringing about new implications for its possible roles in structural formation and remodeling of bone.Since its invention, operation of AFM in liquid for imaging soft biomaterials has been hindered by a low quality factor caused by large drag forces on the cantilever. Utilizing the small dimensions of a nanoneedle, the new method presented in this dissertation resolves the complications by keeping the cantilever outside of the liquid and using a nanoneedle attached to the AFM probe as the sensing element in liquid. It was shown that this method in liquid maintained the harmonic dynamic characteristics of the cantilever similar to that in air and had an intrinsic high quality factor. The performance of the new method is demonstrated through imaging single collagen fibrils in liquid as well as the extremely soft membrane of living cells under physiological conditions.
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Scanning Probe Microscopy of Biomaterials and Nanoscale Biomechanics