Single Molecule Imaging Methods and Novel Computational Motion Analysis used to Characterize the Transitions of Single Molecules between Multiple States of Motion: Determining the Biochemical Kinetics of Single Molecules in In Vitro and In Vivo Systems

Abstract

In recent years, the field of biophysics has placed an increasing emphasis on characterizing single molecule motion as a tool to understand the complex biochemistry of experimental systems such as molecular motors which convert chemical energy to mechanical force. The interpretation of these motions remains limited because single molecules rarely display a single type of motion and instead dynamically switch between many different states of motion as they change biochemical state and interact with their local environment. This dynamic switching complicates the analysis of single molecule motion because the classical motion equations assume homogenous behavior preventing their direct application to switching trajectories. In this dissertation we develop a novel non-averaging displacement analysis (NADA, Chapter 2) that graphically represents all of the motion within single molecule trajectories at once allowing the different states of motion to segregate into different populations where their mechanical properties and lifetimes can be measured. We then applied our NADA method to analyze the motion of individual regulated thin filaments to provide insights into the basic mechanisms of striated muscle regulation (Chapter 3). In the final chapter we described the software developed to perform the analysis within the dissertation including extracting positional information from video images of fluorescently labeled molecules with intensity profile based positional refinement at high temporal resolution, curating the measured trajectories and performing the MSD and NADA methods on the measured trajectories (Chapter 4).