Fluorescence correlation spectroscopy with sub-diffraction-limited resolution using near-field optical probes

Abstract

We report fluorescence correlation spectroscopy (FCS) measurements using near-field scanning optical microscopy (NSOM) probes to produce a sub-diffraction-limited observation area. An order of magnitude reduction in the area compared to confocal FCS has been achieved. We also demonstrate a simple means to model the autocorrelation decay due to diffusion within the excitation profile at the 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.