| dc.description.abstract | Many motile bacteria are driven by the bacterial flagellar motor (BFM). The BFM consists of a rotor, a stator, and a filament through a self-assembly mechanism. The BFM is powered by a specific ion motive force (IMF) that generates torque and propulsion for the bacteria. Also, a BFM is a rotational switchable two-state motor. The rotor switches between counterclockwise/clockwise (CCW/CW) rotation when intracellular chemotaxis signaling proteins bind/unbind to the rotor of the BFM (C-ring). As a result, bacteria can overcome thermal fluctuations and navigate a wide range of environments.
In this thesis, we aim to study the motility and chemotaxis of lophotrichous bacteria. We have developed a 3-dimensional tracking method and real-time visualization of flagella during swimming. In phase-contrast 3D tracking, we introduce spherical distortion into phase-contrast microscopy to induce the point spread function (PSF) asymmetry on the z-axis. The z-position of bacteria can be mapped by computing the 2D cross-correlation between the target image and the pre-labeled depth database. Therefore, we can reconstruct spatial trajectory information. However, standard optical microscopes cannot easily observe these 20 nm thin flagellar filaments due to the low scattering cross-section. Therefore, we take the advantage of sheathed flagella that is contiguous with the outer membrane for rapid fluorescence labeling by the lipophilic dye. To capture the flagellar filament configuration during the swimming at several hundred Hz rotations in the wild field of view, we developed the strobe lighting method. We integrate and synchronize cameras and lights through NI-DAQ. Send a series of pulse-width modulation (PWM) signals through NI-DAQ to trigger the devices simultaneously. Thus, we can achieve the imaging of a large field of view (> 1024 pixels) at low frame rates (< 100 Hz) with clear rotating flagellar filament configuration within 200 microseconds (> 5000 Hz).
Known swimming patterns are run-and-tumble in Escherichia coli, run-reverse in Vibrio alginolyticus, and push-wrap in some bacteria. Different swimming patterns allow bacteria to efficiently navigate various environments. We chose A. fischeri, which is a lophotrichous system, as our study system. We discovered two alternative speeds for forward and backward motion through phase-contrast 3D tracking. Then, A. fischeri faced low- and high-viscosity environments using push-pull and push-wrap modes. In addition, A. fischeri synchronizes motor switching and polymorphic changes to prolong backward duration. Finally, we constructed a complete swimming pattern of A. fischeri by fluorescence strobe illumination microscopy. Our simulations then showed that prolonged backward run time enables efficient chemotactic navigation at high viscosity. | en_US |