Development and Applications of Highly Multiplexed Super-Resolution Microscopy

Single-molecule localization based super-resolution imaging methods have seen considerabledevelopment since their inception over a decade ago. Such advances have enabled countlessbiological discoveries and have positioned the field of single-molecule localization microscopy(SMLM) at the forefront of biological research. This dissertation describes the expansion ofconventional SMLM methodologies to facilitate multicolor, true-color, and correlative imaging,and details specific applications for each of these developments. First, we utilize a split-emissionoptical setup to enable facile multicolor super-resolution imaging of 3 or more fluorophores.Then, we develop a novel technique in which a dispersive prism is used to obtain the entirefluorescence spectra of millions of single molecules. Finally, we apply nonrigid imageregistration techniques to enable the correlative imaging of live and fixed Drosophila larvae. Wethen describe the application of advanced multicolor imaging methods to the study offundamental biological processes. These include the discovery of a novel organization ofmembrane ion channels in sperm cells, the identification of proteins responsible for forcegeneration in mitotic spindles, and the characterization of new structure-function relationships invesicular transport pathways such as COPII protein trafficking and autophagy