Browsing by Subject "Photonic force microscope"
Now showing 1 - 4 of 4
- Results Per Page
- Sort Options
Item Development of quantitative three-dimensional thermal noise imaging of biopolymer filaments(2012-05) Kochanczyk, Martin David; Florin, Ernst-Ludwig; Shubeita, George; Marder, Michael; Bengtson, Roger; Zhang, John XBiopolymer 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.Item Measuring the nonconservative force field in an optical trap and imaging biopolymer networks with Brownian motion(2011-12) Thrasher, Pinyu Wu; Florin, Ernst-LudwigOptical tweezers have been widely used by biophysicists to measure forces in single molecular processes, such as the force of a motor molecule walking and the force of a DNA molecule winding and unwinding. In these and similar force measurements, the usual assumption is that the force applied to a particle inside the tweezers is proportional to the displacement of the particle away from the trap center like Hookean springs, which would imply that the force field is conservative. However, the Gaussian beam model has indicated that the force field generated by optical tweezers is actually nonconservative, yet no experiments have measured or accounted for this effect. We introduce an experimental method -- the local drift method -- that can measure the force field in optical tweezers with high precision without any assumptions about the functional form of the force field. The force field is determined by analyzing the Brownian motion of a trapped particle. We successfully applied this method to different sizes of particles and measured the three dimensional force field with 10 nm spatial resolution and femtonewton precision in force. We find that the force field is indeed nonconservative. The nonconservative contribution increases radially away from the optical axis for both small and large particles. The curl vector field -- a measurement of the nonconservative force field -- reverses direction from counter-clockwise for small particles in the Rayleigh regime to clockwise for large particles in the ray optics regime, consistent with the different scattering force profiles in the two distinct scattering regimes. Together with the thermal fluctuations of the trapped particle, the nonconservative force can cause a complex flux of energy into the system. Optically-confined Brownian motion is further used to probe nanostructures such as a biopolymer network. This technique -- thermal noise imaging -- uses a Brownian particle as a "natural scanner" to explore a biopolymer network by moving the Brownian particle through the network with optical tweezers. The position fluctuations of the probe particle reflect the location of individual filaments as excluded volumes. The resolution of thermal noise imaging is directly coupled to the size of the probe particle. A smaller probe is capable of exploring smaller pore sizes formed by dense network. Previously, a 200 nm polystyrene particle had been used to probe an agar network. In this work, 100 nm gold probe particles are used to enhance the resolution. A 100 nm particle explore a network with mesh 2³ times smaller and therefore enhance the network resolution by 2³ times. A 100 nm particle also improves the imaging speed by a factor of 2 because of its faster diffusion. Three-dimensional thermal noise images of agarose filaments are obtained and a resolution of 10 nm for the position of the filaments is achieved. In addition, a gold particle is trapped with significantly less power than a polystyrene particle of the same size, indicating the possibility for using even smaller gold particles to further improve the resolution.Item Optical trap based studies of single, bundled, and cross-linked microtubules(2021-08-06) Losowyj, Daniel Joseph; Florin, Ernst-Ludwig; Marder, Michael P; Gordon, Vernita D; Baker, AaronCytoskeletal networks within cells provide structural support and fulfill crucial biological roles for eukaryotic organisms. These networks display emergent behaviors like highly nonlinear elasticity that originate in the properties of individual filaments and the three-dimensional architecture of the network. In particular, the connectivity between filaments has major implications for the stress distribution and mechanics of the network, which, in combination with the stiffness of the constituent filaments, determine the magnitude of thermal fluctuations within the network. Beyond their structural importance, cytoskeletal networks also serve as highways for intracellular transport while simultaneously acting as obstructions that hinder diffusive motion on the nanoscopic scale. The immense difficulty associated with studying these networks from a single filament up to a collection of cross-linked filaments means that there is a dearth of experimental data in this area. Here, we fill this gap with two novel methods of studying cytoskeletal filaments focusing specifically on microtubules, semiflexible filaments with rich behavior on the single filament level and network properties that depart from those of other cytoskeletal networks. First, we use thermal noise imaging, a high bandwidth, probe-based technique, to observe cross-linked networks of microtubules. New analysis methods allow us to localize individual filaments with 2.5 nm precision, one-tenth of the diameter of a microtubule, and additionally to track filaments’ positions through intersections in the network, which have thwarted localization attempts in previous studies. This leads to the first direct measurement of a cross-link between connected filaments with a distance of just 1.4 nm found to be separating the microtubules. Furthermore, observations of filament fluctuations show that they are directly connected to the network architecture, suggesting that tensions within the network caused by cross-links may be quantified by these measurements. Diffusion of a spherical particle is also tracked throughout these experiments so that spatial variations in the diffusion constant can be mapped with a resolution of 10 nm. These measurements show that single microtubules can dramatically influence the motion of our probe particle, and, since the probe is similar in size to vesicles within cells, microtubules may play a significant role in the intracellular diffusion of such vesicles and other biologically important particles. After analyzing cross-linked networks, we then present the standing wave optical trap (SWOT) as a means of manipulating individual filaments with the potential to craft custom networks of microtubules. We show that not only can the SWOT trap a single microtubule, it can also manipulate microtubule bundles and individual filaments through the polarization of the trapping laser. This surprising degree of experimental control can only be explained if microtubules have a birefringence of at least 0.02, almost ten times larger than previously thought, opening the door to new possibilities for crafting designer networks filament by filament.Item Submicroscopic characterization of biopolymer networks in solution by Thermal Noise Imaging(2013-05) Bartsch, Tobias Fabian; Florin, Ernst-Ludwig; Shubeita, George T; Aldrich, Richard W; Demkov, Alex A; Fink, ManfredBiopolymer networks display a wide range of interesting mechanical properties that are essential for living organisms. For example, a highly nonlinear elastic response to strain gives biopolymer networks the ability to comply with small stresses but to resist large ones. These macroscopic mechanical properties have their origin in the properties of the individual filaments and their connectedness, like cross-linking geometry and pore size distribution. While the macroscopic properties of biopolymer networks have been extensively studied, there has been a lack of experimental techniques that can simultaneously determine mechanical and architectural properties of networks in situ with single filament resolution. This work introduces Thermal Noise Imaging (TNI) as a novel quantitative method to address these issues. TNI is a three-dimensional scanning probe technique that utilizes the confined thermal motion of an optically trapped particle as a three-dimensional, noninvasive scanner for soft, biological material. Using a photonic force microscope (PFM) custom built for this research, the position of the probe can be detected with nanometer precision and megahertz bandwidth. Two sets of single molecule experiments are described that demonstrate the microscope's exceptional precision and stability. Micrometer scale thermal noise images inside a collagen network are shown and quantitative information about cross-linking geometry is extracted from the data. Further, by imaging microtubules grafted to a support it is shown that the acquired data yield information about the transversal fluctuations of the imaged fibers and about fiber elasticity. These results pave the way for an investigation of force distributions inside biopolymer networks on the single filament level.