Simultaneous two‐photon fluorescence correlation spectroscopy and lifetime imaging of dye molecules in submicrometer fluidic structures

Fluorescence correlation spectroscopy (FCS) is a very sensitive technique that can be used, e.g., for the measurement of low concentrations and for the investigation of transport of fluorescent molecules. Fluorescence lifetime imaging (FLIM) provides spatially resolved information about molecular fluorescence lifetimes reflecting the interactions of the molecules with their environment. We have applied simultaneous two-photon FCS and FLIM to probe the behavior of fluorescent molecules diffusing in submicrometer silicon oxide channels. Our measurements reveal differences in fluorescence lifetimes compared to bulk solution that result from the effects of confinement and the presence of interfaces. Confinement also affects diffusional characteristics of fluorophores as reflected in fluorescence autocorrelation functions. These possible consequences of both spatial confinement and the presence of interfaces between media with different refractive indices on the diffusion and fluorescence lifetime of molecules in nanostructures are discussed in general.