Issue 1, 2013

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

Abstract

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.

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

Supplementary files

Article information

Article type
Edge Article
Submitted
25 iyl 2012
Accepted
05 okt 2012
First published
05 okt 2012

Chem. Sci., 2013,4, 220-225

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

M. K. Lee, J. Williams, R. J. Twieg, J. Rao and W. E. Moerner, Chem. Sci., 2013, 4, 220 DOI: 10.1039/C2SC21074F

To request permission to reproduce material from this article, please go to the Copyright Clearance Center request page.

If you are an author contributing to an RSC publication, you do not need to request permission provided correct acknowledgement is given.

If you are the author of this article, you do not need to request permission to reproduce figures and diagrams provided correct acknowledgement is given. If you want to reproduce the whole article in a third-party publication (excluding your thesis/dissertation for which permission is not required) please go to the Copyright Clearance Center request page.

Read more about how to correctly acknowledge RSC content.

Social activity

Spotlight

Advertisements