Spatially Resolved Scattering Correlation Spectroscopy Using a Total Internal Reflection Configuration

In the paper, we present a novel single particle method, named spatially resolved scattering correlation spectroscopy (SRSCS), based on a total internal reflection (TIR) configuration and strong resonance light scattering (RLS) of silver nanoparticles (AgNPs). The principle of SRSCS is similar to fluorescence correlation spectroscopy (FCS), and it is based on measuring the RLS fluctuations in a small volume due to Brownian motion of single nanoparticles. We first established a highly sensitive SRSCS system. In the SRSCS system, a millimeter-scale hole is employed to efficiently separate nanoparticle scattering light from the background reflected beam, and an electron multiplying charge-coupled device (EMCCD) is used as an array detector. The SRSCS system was successfully used for detection and imaging of single AgNPs in solution. Furthermore, we developed the model of SRSCS according to the FCS method and systematically investigated the effects of certain factors such as particle concentration, viscosity of the solution, hardware and software binning and accumulation time on SRSCS measurements using AgNPs as a model sample. A series of calibration experiments were conducted, and the experimental data obtained were in good agreement with the SRSCS model. This new method is multiplexing, spatially resolved, and free of photobleaching and may become a useful method for study on heterogeneous systems, such as the motion of proteins on the cell membrane