Enzymatic activation of nitro-aryl fluorogens in live bacterial cells for enzymatic turnover-activated localization microscopy

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...

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Bibliographic Details
Published in:Chemical science (Cambridge) 2013-01, Vol.42 (1), p.220-225
Main Authors: Lee, Marissa K, Williams, Jarrod, Twieg, Robert J, Rao, Jianghong, Moerner, W E
Format: Article
Language:English
Subjects:
Bacteria
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Summary: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.
ISSN:2041-6520
2041-6539
DOI:10.1039/c2sc21074f