Zero-Mode Waveguides: Subwavelength optical apertures for single molecule studies at high molecular concentrations

The study of the individual components of biological systems requires high spatial and temporal resolution at physiologically-relevant concentrations. Although many optical techniques have enabled the study of single biomolecules, these techniques are inherently restricted by the diffraction limit. This in turn limits the available range of molecular concentrations that can be studied at the single molecule level to the nano or picomolar regimes. Thus, alternative techniques for the confinement of excitation volumes to sub- diffraction limit dimensions have been eagerly sought. We have approached this problem through the use of nanostructured substrates. Zero-mode waveguides (ZMWs), sub-wavelength optical apertures fabricated in a thin aluminum film, are devices which confine optical excitation to atto- or zeptoliter volumes. Such confinement allows the study of diffusion processes at micro- to millimolar concentrations, which are physiologically relevant in biological systems. In this presentation, applications of such devices to the study of biomembranes will be discussed. In combination with fluorescence correlation spectroscopy, the diffusive behavior of individual lipids, receptors and membrane bound proteins is characterized within ZMWs. The nanoapertures were also applied to the study of various cellular membrane components, demonstrating the possibility of using such excitation volume confinement in the study of membrane components in live, environmentally responsive cells. It is proposed that ZMWs will be useful devices in the study of biomolecular processes where the diffusion and association of individual molecules needs to be monitored. Furthermore, they could aid in the development of optical biosensors where single molecule resolution is warranted.