Optimization of Modular, Long-Range, Ultra-Fast Optical Tweezers With Fluorescence Capabilities for Single-Molecule and Single-Cell Based Biophysical Measurements

Author

Subash C. GodarFollow

Date of Award

5-2022

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Physics and Astronomy

Committee Chair/Advisor

Joshua Alper

Committee Member

Emil Alexov

Committee Member

Hugo Sanabria

Committee Member

Sean Brittain

Abstract

An Optical tweezer is a tightly focused laser beam that applies and senses precise and localized optical force to a dielectric microsphere and offers a unique and effective tool for manipulating the single cell or cell components, including nucleotides and dynein motor proteins. Here, I used highly stabilized optomechanical components and ultra-sensitive detection modules to significantly improve the measurement capabilities over a wide range of temporal and spatial scales. I combined the optical tweezer-based force spectroscopy technique with fluorescence microscopy to develop an integrated high-resolution force-fluorescence system capable of measuring displacements at sub-nanometer, forces at sub-piconewton over a temporal range of milliseconds to 10s of seconds. I developed a flow cell-based high throughput DNA tether assay to probe the correlation between the structure, dynamics, and functionality of Thiamine pyrophosphate binding RNA Riboswitch. I used this dynamic single-molecule assay to analyze the metabolite-dependent dynamic switching behavior of the riboswitch. Furthermore, I used a high throughput single-cell trapping assay in combination with high-speed video microscopy to characterize the dynein regulatory role of light chain-2 in dynein-based flagellar motility in the Trypanosoma brucei, a parasitic protozoan. Finally, I used the fluorescence capability of the system to visualize the fluorescently labeled microtubules and study the molecular interactions between the dynein motor protein and microtubule in either the single-molecule unbinding assay performed using mouse cytoplasmic dynein MTBD or bulk protein gliding assay performed using the Chlamydomonas or Trypanosome flagellar dyneins. I expect understanding molecular interactions provides a platform for their use as novel therapeutic targets.

Recommended Citation

Godar, Subash C., "Optimization of Modular, Long-Range, Ultra-Fast Optical Tweezers With Fluorescence Capabilities for Single-Molecule and Single-Cell Based Biophysical Measurements" (2022). All Dissertations. 2995.
https://open.clemson.edu/all_dissertations/2995