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Pure Appl. Chem., 2009, Vol. 81, No. 9, pp. 1645-1653

Published online 2009-08-10

Near-field optical probes provide subdiffraction-limited excitation areas for fluorescence correlation spectroscopy on membranes

Dusan Vobornik1, Daniel S. Banks2, Zhengfang Lu1, Cécile Fradin2, Rod Taylor1 and Linda J. Johnston1*

1 Steacie Institute for Molecular Sciences, National Research Council of Canada, Ottawa, Ontario K1A 0R6, Canada
2 Department of Physics and Astronomy, McMaster University, Hamilton, Ontario L8S 4M1, Canada

Abstract: Near-field optical probes have been used to produce a subdiffraction-limited observation area for fluorescence correlation spectroscopy (FCS) experiments on supported membranes. The design of a bent, etched fiber probe that is compatible with biological imaging in an aqueous environment is described. This probe design is used for proof of principle experiments to measure lipid diffusion in a fluid-supported bilayer. A reduction in excitation area of approximately one order of magnitude (relative to a confocal FCS experiment) is obtained with a probe aperture diameter of 140 nm. We also demonstrate a simple approach for modeling the autocorrelation decay due to diffusion within the excitation profile at the near-field scanning optical microscopy (NSOM) probe aperture. The use of probes with smaller apertures is expected to provide an additional order of magnitude reduction in the observation area, thus enabling the study of cellular membranes with higher concentrations of fluorophores than is currently possible with diffraction-limited techniques.