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Development and Applications of Highly Multiplexed Super-Resolution Microscopy


Single-molecule localization based super-resolution imaging methods have seen considerable

development since their inception over a decade ago. Such advances have enabled countless

biological discoveries and have positioned the field of single-molecule localization microscopy

(SMLM) at the forefront of biological research. This dissertation describes the expansion of

conventional SMLM methodologies to facilitate multicolor, true-color, and correlative imaging,

and details specific applications for each of these developments. First, we utilize a split-emission

optical 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 entire

fluorescence spectra of millions of single molecules. Finally, we apply nonrigid image

registration techniques to enable the correlative imaging of live and fixed Drosophila larvae. We

then describe the application of advanced multicolor imaging methods to the study of

fundamental biological processes. These include the discovery of a novel organization of

membrane ion channels in sperm cells, the identification of proteins responsible for force

generation in mitotic spindles, and the characterization of new structure-function relationships in

vesicular transport pathways such as COPII protein trafficking and autophagy.

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