Mancebo Jr, Angel2025-03-212025-03-212022-12https://hdl.handle.net/11299/270590University of Minnesota Ph.D. dissertation. December 2022. Major: Physics. Advisor: Elias Puchner. 1 computer file (PDF); x, 176 pages.Single-molecule localization microscopy (SMLM) is a powerful technique that enables the observation of protein organization in living cells at a resolution well below the optical diffraction limit. Through the wide variety of specific labels that are available, it has become possible to use two-color SMLM to observe and quantify colocalization between two protein species. The information obtained from their colocalization can yield insights into their possible interaction as well as their relative enrichment. Identifying sites of interaction remains a challenge, in part because there are often large concentration ratios between interacting partners and few interactions occurring relative to the number of the more abundant species. The robust and genetically-manipulable budding yeast, Saccharomyces cerevisiae, with many genes and pathways homologous to those in mammals, serves as a powerful tool for expediting studies in eukaryotic cells. However, two-color SMLM has not been possible in living yeast. This is because the dyes that are used in mammalian cells for achieving a second color are actively exported by yeast. In this thesis I present work towards the development of two-color SMLM in living yeast. I show a simple approach for patterning of photoactivation light with sub-diffraction precision, which has the added benefit of reducing phototoxicity. This patterned photoactivation also enables diffusion contrast photoactivation localization microscopy (dcPALM), which exploits differences in mobility between proteins that are interacting or not interacting with a binding partner to reduce the non-interacting background. Progressing closer into two-color SMLM, I show a method for efficiently separating localizations based on cross-correlation between two channels, with an application to a mammalian system in which it was demonstrated that a critical number of a key protein in the initiation of autophagy needed to be present to initiate autophagy. Finally, I show for the first time two-color SMLM in living yeast — particularly with Janelia Fluor HaloTag dyes — and the characterization of a drug that facilitates the uptake of the dyes into yeast.encross-correlationdigital micromirror devicefluorescence microscopyphotoactivation localization microscopysingle-molecule localization microscopyyeastThesis or Dissertation