Fluorescence Characterization Studies for Single Molecules Isolated in Hybrid Zero-Mode Waveguides

W.E. Martin
University of Kentucky,
United States

Keywords: zero-mode waveguide, fluorescence, single, molecule, plasmonic, nanostructure

Summary:

Single-molecule fluorescence spectroscopy has become increasingly popular in the field of biological imaging due to techniques that can probe beyond the normal diffraction limit of light; although such techniques are limited to highly efficient fluorophores. Zero-mode waveguides (ZMWs) are nanoscale plasmonic apertures milled into thin metal films, which have shown the ability to enhance fluorescence signals. These apertures are strongly dependent on metallic composition, size (aperture diameter), and in the case of single molecule studies, the spatial position of molecules in relation to the outer metal edges of the aperture. Single fluorophores are used as probes to gauge how these apertures can manipulate photophysical properties. Characterization studies have been performed by probing fluorophores both in solution1 and by isolating them to the base of an aperture2. These studies have provided key insights regarding which composition/size combinations are best suited for specific fluorophores and which should be avoided. We present novel fabrication techniques for hybrid ZMW structures and probe their effects in various regions of the visible spectrum. Our pulsed excitation confocal microscope setup allows us to probe changes in both the excitation and emission pathways of isolated single fluorophores within the hybrid ZMWs. These structures should play a major role in the increased tunability of ZMWs for biological use at the single molecule level. (1) Aouani, H.; Hostein, R.; Mahboub, O.; Devaux, E.; Rigneault, H.; Ebbesen, T. W.; Wenger, J. Saturated excitation of fluorescence to quantify excitation enhancement in aperture antennas. Optics express 2012, 20, 18085-18090. (2) Holzmeister, P.; Acuna, G. P.; Grohmann, D.; Tinnefeld, P. Breaking the concentration limit of optical single-molecule detection. Chemical Society Reviews 2014, 43, 1014-1028.