Date of Award
Summer 8-22-2026
Document Type
Masters Thesis
Degree Name
M.S. in Biological Sciences
Organizational Unit
College of Natural Science and Mathematics, Biological Sciences
First Advisor
Schuyler van Engelenburg
Second Advisor
Scott Barbee
Third Advisor
Joseph Angleson
Fourth Advisor
Brian Michel
Copyright Statement / License for Reuse
All Rights Reserved.
Keywords
Single-molecule localization microscopy (SMLM), Direct stochastic optical reconstruction microscopy (dSTORM), Oxygen scavenging system, Recombinant protein engineering, Pyranose oxidase, APEX2, Superresolution microscopy, Fluorescence microscopy
Abstract
Single-molecule localization microscopy (SMLM) relies on enzymatic oxygen scavenging systems to suppress photobleaching and promote fluorophore photoswitching. Conventional systems depend on commercially sourced enzymes, motivating development of sustainable recombinant alternatives. This work investigated two recombinant oxygen scavenging strategies for direct stochastic optical reconstruction microscopy (dSTORM). Recombinant protocatechuate 3,4-dioxygenase (PCD)was expressed and purified but exhibited poor batch-to-batch reproducibility and limited long-term stability despite extensive biochemical optimization. These findings established reproducibility and storage stability as essential engineering requirements. Building on these observations, a fully recombinant pyranose oxidase/ascorbate peroxidase (PyOx/APEX2) system demonstrated imaging performance comparable to conventional glucose oxidase/catalase and commercial PyOx/APEX2 buffers while using substantially lower PyOx concentrations. The recombinant system exhibited robust reproducibility and supported quantitative dSTORM imaging of nuclear pore complexes and HIV-1 conical cores. Collectively, this work establishes recombinant oxygen scavenging as a practical, sustainable, and engineerable approach for superresolution microscopy.
Copyright Date
8-2026
Publication Statement
Copyright is held by the author. User is responsible for all copyright compliance.
Rights Holder
Olyvia Weaver
Provenance
Received from ProQuest
File Format
application/pdf
Language
English (eng)
Extent
121 pgs
File Size
74 MB
Recommended Citation
Weaver, Olyvia, "Development of Sustainable Recombinant Oxygen Scavenging Systems for Single-Molecule Superresolution Microscopy" (2026). Electronic Theses and Dissertations. 2812.
https://digitalcommons.du.edu/etd/2812