Nanoscience and Microsystems ETDs

Publication Date

Summer 7-29-2025

Abstract

Super-resolution techniques developed over the past couple of decades enabled us to bypass the classical diffraction limit of light by exploiting the independent behavior of the fluorophores labeling the cellular sample. In localization-based super-resolution approaches, sparse activation of fluorophores allows precise localization of individual emitters, enabling super-resolution image reconstruction and quantitative analysis of sub cellular structures within 10 to 200 nm range. This dissertation covers various developments and applications of single molecule localization microscopy (SMLM) methods. The MATLAB Instrument Control (MIC) package enables automated data collection on custom-built microscopes, while Single Molecule Imaging Toolbox Extraordinaire (SMITE) facilitates the analysis of fluorescence single-molecule imaging data, focusing on SMLM and single-particle tracking (SPT). Two applications of a custom-built Sequential super-resolution microscope are explored: investigating how mammalian ATG8 (mATG8) proteins regulate the permeability of autophagic membranes and estimating the field dependent aberrations directly from SMLM blinking data across a large field-of-view (FOV). Next, we develop a novel approach to Supercritical Angle Localization Microscopy (SALM), leveraging point spread function (PSF) modeling in a simplified, single channel configuration. In this approach, PSF modeling method combined with the stage-tilt correction algorithm improves the axial localization precision in 3D SMLM. In the end, we discuss the Super Localization via Image inVERsion interferometry (SLIVER) technique that allows near quantum-optimal precision in separation estimation between two incoherent point sources. A Total Internal Reflection Fluorescence (TIRF) microscope is built to integrate with the SLIVER setup for direct detection imaging and centroid estimation of closely spaced fluorescent beads.

Keywords

Super-resolution microscopy, Single molecule localization microscopy (SMLM), SMLM instrument control and data analysis, Autophagy, Large field-of-view SMLM imaging, Quantum-optimal imaging

Document Type

Dissertation

Language

English

Degree Name

Nanoscience and Microsystems

Level of Degree

Doctoral

Department Name

Nanoscience and Microsystems

First Committee Member (Chair)

Keith A. Lidke

Second Committee Member

Francisco E. Becerra

Third Committee Member

Diane S. Lidke

Fourth Committee Member

Matthew R. Lakin

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Available for download on Thursday, July 29, 2027

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