Adhikari, Santosh2023-03-272023-03-272022-06https://hdl.handle.net/11299/253430University of Minnesota Ph.D. dissertation. June 2022. Major: Physics. Advisor: Elias Puchner. 1 computer file (PDF); viii, 161 pages.Quantitative Single-Molecule Localization Microscopy (SMLM) is evolving into a powerful biophysical technique to study biological processes below the optical diffraction limit. However, due to the motion of the imaged structures during the long data acquisition times, most studies have been limited to fixed cells. In addition, it has been a challenge to label hydrophobic environments such as lipid droplets (LDs) due to the properties of available fluorophores. LDs are highly dynamic and actively regulated lipid and energy storage organelle of a cell that is essential to maintain lipid and energy homeostasis in the cell during metabolic changes and to buffer the fatty acid levels to protect the cell from lipotoxicity. Cells tightly control the number, size, and spatial distribution of LDs by regulating the density of functional enzymes on the LD surface. In this thesis, I present the application of quantitative SMLM to lipid droplet biology to reveal how yeast cells dynamically regulate the density of fatty acid activating enzymes on their lipid droplets. This density regulation enables cells to respond to the energy and lipid demands during metabolic changes. I develop a novel mechanism of conventional BODIPY conjugates that enables quantitative SMLM imaging of fatty acid and neutral lipid distributions with two colors in living yeast and mammalian cells. The obtained high resolution and single-molecule sensitivity of these measurements allowed us to gain new biological insights into fatty acid trafficking and metabolism. Finally, I will present the development of motion-corrected photo-activated localization microscopy (mcPALM) for imaging moving structures with super-resolution and show the potential for new insights into the structure and dynamics of endosomes and telomeres in living yeast and mammalian cellsenBODIPY dyesFluoresenceLipid dropletsSingle moleculeSuper-resolutionTrackingThesis or Dissertation