Optical microscopy has successfully been used to visualize the dynamics of living systems for decades. Numerous microscopic techniques, including single particle tracking (SPT), have been developed to measure dynamic processes in living cells with minimum disruption to the cellular functions. SPT is capable of accessing individual behaviors of imaging probe (single molecule or nanoparticle) with high spatial and temporal resolution. It has been utilized for investigating dynamic events of single molecule and nanoparticle in many different biological systems. Single particle orientation and rotational tracking (SPORT) studies not only the spatial movements as in conventional SPT but also the orientation and rotational behavior of imaging probes in order to reveal the molecular mechanisms involved in fundamental motions. A variety of experimental techniques have been reported for determining the orientation and rotational motions of optical imaging probes. Differential interference contrast (DIC) microscopy, along with the use of anisotropic plasmonic nanoparticles as optical probes, offers unique ability in SPORT study.

Synthesis of novel imaging probes, innovations in optical implementation, advance in data analysis could all contribute to the development and expanding the applications of SPORT techniques in dynamic studies. Multishell Au/Ag/SiO2 core-shell hybrid nanorods with tunable optical properties were synthesized and used as new SPORT probes in DIC microscopy. These nanorods provided enhanced detection sensitivity, improved stability and additional surface modification possibility. The addition of a wedge prism and the implementation of auto-focusing algorithm formed Parallax-DIC microscopy in the 5D-SPT method. It enables the simultaneous 3D spatial tracking and orientation determination in visualization of intracellular transport of cargos in live cells. Autocorrelation function analysis and binning function in imageJ was utilized for DIC data processing. Experimental parameters, such as trajectory length of the SPT tracking and frame rate (exposure time) were investigated for the rotation of surface modified gold nanorods on synthetic lipid bilayers with assistance of computer simulations. However, much effort is still required in exploring new imaging probes, optical microscopic implementations, and data analysis methods to further extend the potential of DIC-based SPORT techniques in dynamic studies.