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Addressing the Requirements of High‐Sensitivity Single‐Molecule Imaging of Low‐Copy‐Number Proteins in Bacteria
Author(s) -
Tuson Hannah H.,
Aliaj Alisa,
Brandes Eileen R.,
Simmons Lyle A.,
Biteen Julie S.
Publication year - 2016
Publication title -
chemphyschem
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.016
H-Index - 140
eISSN - 1439-7641
pISSN - 1439-4235
DOI - 10.1002/cphc.201600035
Subject(s) - bacillus subtilis , fluorescence , fluorescence microscope , single molecule experiment , fluorescence lifetime imaging microscopy , escherichia coli , biophysics , molecule , resolution (logic) , microscopy , chemistry , bacteria , microscope , biology , biochemistry , physics , optics , gene , genetics , organic chemistry , artificial intelligence , computer science
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.