title: Simultaneous Surface-Near and Solution Fluorescence Correlation Spectroscopy.
abstract: We report the first simultaneous measurement of surface-confined and solution fluorescence correlation spectroscopy (FCS). We use an optical configuration for tightly focused excitation and separate detection of light emitted below (undercritical angle fluorescence, UAF) and above (supercritical angle fluorescence, SAF) the critical angle of total internal reflection of the coverslip/sample interface. This creates two laterally coincident detection volumes which differ in their axial extent. While detection of far-field UAF emission producesa standard confocal volume, near-field-mediated SAF produces a highly surface-confined detection volume at the coverslip/sample interface which extends only ~200 nm into the sample. A characterization of the two detection volumes by FCS of free diffusion is presented and compared with analytical models and simulations. The presented FCS technique allows to determine bulk solution concentrations and surface-near concentrations at the same time.
authors.affiliation: Randall Division of Cell and Molecular Biophysics, King's College London, London, SE1 1UL, UK. christian.winterflood@kcl.ac.uk.
authors.forename: Christian M
authors.identifier: http://orcid.org/0000-0001-5621-3695
authors.initials: CM
authors.lastname: Winterflood
authors.affiliation: Department of Chemistry, University of Zurich, Winterthurerstrasse 190, 8057, Zurich, Switzerland.
authors.forename: Stefan
authors.initials: S
authors.lastname: Seeger
keywords: Far-field; Fluorescence correlation spectroscopy; Near-field; Supercritical angle fluorescence; Surface-selective; Undercritical angle fluorescence