title: Addressing the Requirements of High-Sensitivity Single-Molecule Imaging of Low-Copy-Number Proteins in Bacteria.
abstract: Single-molecule fluorescence super-resolution imaging and tracking provide nanometer-scale information about subcellular protein positions and dynamics. These single-molecule imaging experiments can be very powerful, but they are best suited to high-copy number proteins where many measurements can be made sequentially in each cell. We describe artifacts associated with the challenge of imaging a protein expressed in only a few copies per cell. We image live Bacillus subtilis in a fluorescence microscope, and demonstrate that under standard single-molecule imaging conditions, unlabeled B. subtilis cells display punctate red fluorescent spots indistinguishable from the few PAmCherry fluorescent protein single molecules under investigation. All Bacillus species investigated were strongly affected by this artifact, whereas we did not find a significant number of these background sources in two other species we investigated, Enterococcus faecalis and Escherichia coli. With single-molecule resolution, we characterize the number, spatial distribution, and intensities of these impurity spots.
authors.affiliation: Department of Chemistry, University of Michigan, Ann Arbor, MI, 48109, USA.
authors.forename: Hannah H
authors.identifier: 0000-0002-7009-882X
authors.initials: HH
authors.lastname: Tuson
authors.affiliation: Department of Chemistry, University of Michigan, Ann Arbor, MI, 48109, USA.
authors.forename: Alisa
authors.initials: A
authors.lastname: Aliaj
authors.affiliation: Department of Molecular, Cellular, and Developmental Biology, University of Michigan, Ann Arbor, MI, 48109, USA.
authors.forename: Eileen R
authors.initials: ER
authors.lastname: Brandes
authors.affiliation: Department of Molecular, Cellular, and Developmental Biology, University of Michigan, Ann Arbor, MI, 48109, USA.
authors.forename: Lyle A
authors.initials: LA
authors.lastname: Simmons
authors.affiliation: Department of Chemistry, University of Michigan, Ann Arbor, MI, 48109, USA. jsbiteen@umich.edu.
authors.forename: Julie S
authors.initials: JS
authors.lastname: Biteen
mesh_terms: D001412:Bacillus subtilis / Q000378:metabolism*; D001426:Bacterial Proteins / Q000378:metabolism; D013293:Enterococcus faecalis / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism*; D008856:Microscopy, Fluorescence / Q000379:methods*
chemical_list: D001426:Bacterial Proteins
keywords: fluorescence; imaging agents; localization microscopy; single-molecule studies; super-resolution imaging