Article pubs.acs.org/JPCA
A Comparison of Single-Molecule Emission in Aluminum and Gold Zero-Mode Waveguides W. Elliott Martin,† Bernadeta R. Srijanto,‡ C. Patrick Collier,‡ Tom Vosch,§ and Christopher I. Richards*,† †
Department of Chemistry, University of Kentucky, 505 Rose Street, Lexington, Kentucky 40506, United States Center for Nanophase Materials Sciences, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37831, United States § Nano-science Center/Department of Chemistry, University of Copenhagen, Universitetsparken 5, 2100 Copenhagen, Denmark ‡
S Supporting Information *
ABSTRACT: The effect of gold and aluminum zero-mode waveguides (ZMWs) on the brightness of immobilized single emitters was characterized by probing fluorophores that absorb in the green and red regions of the visible spectrum. Aluminum ZMWs enhance the emission of Atto565 fluorophores upon green excitation, but they do not enhance the emission of Atto647N fluorophores upon red excitation. Gold ZMWs increase emission of both fluorophores with Atto647N showing enhancement that is threefold higher than that observed for Atto565. This work indicates that 200 nm gold ZMWs are better suited for singlemolecule fluorescence studies in the red region of the visible spectrum, while aluminum appears more suited for the green region of the visible spectrum.
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INTRODUCTION Single-molecule fluorescence imaging techniques are regularly employed to investigate the heterogeneity of biological systems.1,2 However, despite their many advantages, singlemolecule studies can be hindered by two important factors: limited fluorophore brightness and a need to maintain nanomolar concentrations of fluorescently labeled components.3,4 Fluorescence measurements of single molecules require sensitive detection equipment such as electronmultiplying charge-coupled devices (EMCCDs). The relatively weak signal from single molecules can be easily obscured in the presence of even moderate background fluorescence or cellular autofluorescence5 if one does not want to rely on signal modulation or time-gated techniques.6,7 Therefore, a molecule must emit sufficient photons to be distinguished from the background signal. Fluorophore brightness depends on several factors including molar extinction coefficient, quantum yield of fluorescence and photobleaching, and the length and probability to form intermittent dark states.8 Solution-based single-molecule measurements are typically limited to low concentrations (pM to nM) of fluorescently labeled molecules.3 This is due to the need to limit the number of molecules © XXXX American Chemical Society
present within the focal volume at any given time. This is problematic, because it is often much lower than the physiological concentrations of proteins.3 Zero-mode waveguides (ZMWs) have been used as a tool to address both concentration and brightness issues.9−12 ZMWs consist of nanoscale apertures (
