Single-Molecule Super-Resolution Imaging of Geobacter sulfurreducens under Anaerobic Conditions
Date of Award
Master of Science (MS)
Geobacter sulfurreducens are anaerobic bacteria capable of making electrical contacts with other organisms and extracellular electron acceptors. The challenge of imaging live Geobacter bacteria is maintaining anaerobic conditions during the imaging process. In this thesis, we augment a single-molecule localization microscope (SMLM) with a home-built anaerobic imaging chamber and use constant argon bubbling to maintain oxygen-free imaging conditions. To validate the imaging protocol, we use the transient binding of Nile red to resolve the spherical morphology of lipid-coated glass spheres with nanoscale resolution. However, when imaging Geobacter, the distribution of Nile red localizations is non-uniform, both between different cells on a single coverslip as well as along individual cell membranes. We hypothesize that Geobacter’s ability to transfer electrons extracellularly can reduce single Nile red molecules, thereby transforming them into a dark, non-fluorescent state. We quantitatively compare Nile red activity on wild-type Geobacter to a mutant whose major outer membrane porin-cytochrome complexes are deleted. We demonstrate that SMLM has great potential for quantifying oxidation-reduction activity at the single-molecule level in living bacteria.
Matthew D. Lew
Daniel Bond Michael Vahey
Available for download on Wednesday, May 01, 2024