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.Physic

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Data from: Nanoscopic imaging of thick heterogeneous soft-matter structures in aqueous solution

Precise nanometre-scale imaging of soft structures at room temperature poses a major challenge to any type of microscopy because fast thermal fluctuations lead to significant motion blur if the position of the structure is measured with insufficient bandwidth. Moreover, precise localization is also affected by optical heterogeneities, which lead to deformations in the imaged local geometry, the severity depending on the sample and its thickness. Here we introduce quantitative thermal noise imaging, a three-dimensional scanning probe technique, as a method for imaging soft, optically heterogeneous and porous matter with submicroscopic spatial resolution in aqueous solution. By imaging both individual microtubules and collagen fibrils in a network, we demonstrate that structures can be localized with a precision of ~10 nm and that their local dynamics can be quantified with 50 kHz bandwidth and subnanometre amplitudes. Furthermore, we show how image distortions caused by optically dense structures can be corrected for

Data for Fig 1-4, S4 and S7

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