Date of Award

Summer 8-15-2020

Author's School

McKelvey School of Engineering

Author's Department

Electrical & Systems Engineering

Degree Name

Doctor of Philosophy (PhD)

Degree Type



Single-molecule (SM) fluorescence and its localization are important and versatile tools for understanding and quantifying dynamical nanoscale behavior of nanoparticles and biological systems. By actively controlling the concentration of fluorescent molecules and precisely localizing individual single molecules, it is possible to overcome the classical diffraction limit and achieve 'super-resolution' with image resolution on the order of 10 nanometers.

Single molecules also can be considered as nanoscale sensors since their fluorescence changes in response to their local nanoenvironment. This dissertation discusses extending this SM approach to resolve heterogeneity and dynamics of nanoscale materials and biophysical structures by using positions and orientations of single fluorescent molecules.

I first present an SM approach for resolving spatial variations in the catalytic activity of individual photocatalysts. Quantitative colocalization of chemically triggered molecular probes reveals the role of structural defects on the activity of catalytic nanoparticles. Next, I demonstrate a new engineered optical point spread function (PSF), called the Duo-spot PSF, for SM orientation measurements. This PSF exhibits high sensitivity for estimating orientations of dim fluorescent molecules. This dissertation also discusses a new amyloid imaging method, transient amyloid binding (TAB) microscopy, for studying heterogeneous organization of amyloid structures, which are associated with various aging-related neurodegenerative diseases. Continuous transient binding of dye molecules to amyloid structures generates photon bursts for SM localization over hours to days with minimal photobleaching, yielding about 40% more localizations than standard immunolabeling. Finally, I augment TAB imaging to simultaneously measure positions and orientations of fluorescent molecules bound to amyloid surfaces. This new method, termed single-molecule orientation localization microscopy (SMOLM), robustly and sensitively measures the in-plane (xy) orientations of fluorophores (approximately 9 degree precision in azimuthal angle) near a refractive index interface and reveals structural heterogeneities along amyloid fibrillar networks that cannot be resolved by SM localization alone.


English (en)


Matthew D. Lew

Committee Members

Meredith Jackrel, Ulugbek Kamilov, Joseph O'Sullivan, Quing Zhu,