Phase-Resolved Evanescent Wave Induced Fluorescence. An in Situ Tool for Studying Heterogeneous Interfaces

A new technique that we call phase-resolved evanescent wave induced fluorescence (PREWIF) is introduced and is shown to be useful for studying heterogeneous interfaces under ambient conditions. PREWIF is based on the traditional interfacial selectivity offered by total internal reflection (i.e., the evanescent wave) combined with the picosecond resolving power of phase-resolved fluorescence spectroscopy. Using this new technique we demonstrate the following: (1) the recovery of weak, interfacial fluorescence from a sol-gel-derived thin film doped with a low concentration of fluorophore in the presence of strong Rayleigh scatter; (2) resolution of the individual components in synthetic binary mixtures of fluorophores doped within thin sol-gel-derived films that are coated onto fused silica substrates; (3) elimination of bulk fluorescence and selective monitoring of the interfacial (∼100 Å) species in a model immunosurface; (4) extraction of the individual spectra that contribute to the fluorescence of a fluorescentiy labeled protein physisorbed to a silica surface.