Zero-mode waveguides: Sub-wavelength nanostructures for single molecule studies at high concentrations

The study of single fluorescent molecules allows individual measurements which can reveal characteristics typically obscured by ensemble averages. Yet, single molecule spectroscopy through traditional optical techniques is hindered by the diffraction limit of light. This restricts the accessible concentrations for single molecule experiments to the nano- to picomolar range. Zero-mode waveguides (ZMWs), optical nanostructures fabricated in a thin aluminum film, confine the observation volume to the range of atto- to zeptoliters. Thus, they extend the accessible concentrations for single molecule spectroscopy to the micro- to millimolar regime. Through the combination of ZMWs and fluorescence correlation spectroscopy, a number of biologically relevant systems have been studied at physiological concentrations. In this review, the concept and implementation of ZMWs is outlined, along with their application to the study of freely diffusing, and membrane-bound fluorescent biomolecules.