Journal
CHEMICAL SCIENCE
Volume 4, Issue 1, Pages 220-225Publisher
ROYAL SOC CHEMISTRY
DOI: 10.1039/c2sc21074f
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Funding
- National Institute of General Medical Sciences [R01-GM086196]
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Many modern super-resolution imaging methods based on single-molecule fluorescence require the conversion of a dark fluorogen into a bright emitter to control emitter concentration. We have synthesized and characterized a nitro-aryl fluorogen which can be converted by a nitroreductase enzyme into a bright push-pull red-emitting fluorophore. Synthesis of model compounds and optical spectroscopy identify a hydroxyl-amino derivative as the product fluorophore, which is bright and detectable on the single-molecule level for fluorogens attached to a surface. Solution kinetic analysis shows Michaelis-Menten rate dependence upon both NADH and the fluorogen concentrations as expected. The generation of low concentrations of single-molecule emitters by enzymatic turnovers is used to extract subdiffraction information about localizations of both fluorophores and nitroreductase enzymes in cells. Enzymatic Turnover Activated Localization Microscopy (ETALM) is a complementary mechanism to photoactivation and blinking for controlling the emission of single molecules to image beyond the diffraction limit.
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