Development of quantitative three-dimensional thermal noise imaging of biopolymer filaments

Biopolymer networks perform many essential functions for living cells. Most of these networks show a highly nonlinear mechanical response that is well-studied on the macroscopic scale. While much work has been done to connect the macroscopic responses of networks to microscopic parameters, such as filament stiffness, cross-linking geometry and pore size, there is a lack of experimental techniques that can measure these properties in situ. This thesis presents the development of a quantitative scanning probe imaging technique, which can explore soft matter in an aqueous environment. An optical tweezer-based microscope, called a photonic force microscope, was designed and constructed. A stability analysis method, called Power Spectrum Integration Analysis, was developed and was used to show that the photonic force microscope achieves nanometer precision in the measurement of probe position with a bandwidth of 1MHz. A novel single filament assay was developed that allowed for the isolation and probing of individual biopolymer filaments. A scanning probe technique, called thermal noise imaging, which uses the diffusive motion of an optically trapped nanoparticle as a fast, natural scanner, was used to scan microtubules grafted on one end. The resulting thermal noise images were strongly influenced by the thermally driven, transverse fluctuations of the filaments. Analytical tools, which include Brownian dynamics simulations of probe and filament, were developed to assist quantitative analysis of thermal noise images. The persistence length of individual microtubules was extracted, and the length dependence persistence length for taxol stabilized microtubules was confirmed. The transverse fluctuations of a microtubule grafted on both ends were imaged. Finally, thermal noise images of collagen filaments inside a three-dimensional collagen network were recorded, and variations of the filament diameter were extracted. This thesis establishes thermal noise imaging as a quantitative tool for studying soft material on the nanometer scale, as well as paves the way for investigating force distributions inside biopolymer networks.