Spatial two-photon fluorescence cross-correlation Spectroscopy for controlling molecular transport in microfluidic structures

The increasing availability of microfluidic systems of various geometries and materials for the downscaling of chemical or biochemical processes raises a strong demand for adequate techniques to precisely determine flow parameters and to control fluid and particle manipulation. Of all readout parameters, fluorescence analysis of the fluid or suspended particles is particularly attractive, as it can be employed without mechanical interference and with a sensitivity high enough to detect single molecules in aqueous environments. In this study, we present the determination of flow parameters, such as velocity and direction, in microstructured channels by fluorescence correlation spectroscopy (FCS), a method based on single molecule spectroscopy carried out in confocal optical setups. Different modes of FCS, such as auto- and dual-beam cross-correlation techniques by one- and two-photon excitation, are discussed. Known advantages of two-photon excitation, such as highly restricted detection volumes and low scattering background, are shown to be particularly valuable for measurements in tiny channel systems. Although conventional autocorrelation is sufficient for describing the velocity of single molecules, dual-beam cross-correlation allows the separation of isotropic and anisotropic dynamics, for example, to monitor flow directions or to discriminate against photophysical effects that could be mistaken for mobility parameters. It can be shown that time-gated two-photon excitation in the dual-beam mode significantly lowers the undesired cross-talk between the two measurement volumes. Finally, some applications, such as the calibration of microfluidic sorting units and flow profiling, are demonstrated.