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

Near-field optical probes have been used to produce a subdiffraction-limited ob- servation area for fluorescence correlation spectroscopy (FCS) experiments on supported membranes. The design of a bent, etched fiber probe that is compatible with biological im- aging in an aqueous environment is described. This probe design is used for proof of princi- ple experiments to measure lipid diffusion in a fluid-supported bilayer. A reduction in exci- tation 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 ap- proach 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 concentra- tions of fluorophores than is currently possible with diffraction-limited techniques.