Plasmonic gold nanorods (AuNRs) have been used as orientation probes in optical imaging because of their shape-induced anisotropic optical properties.[1] However, current optical imaging techniques lack the capability to decipher the full three-dimensional (3D) orientation of an infocus gold nanorod in the four quadrants of the cartesian plane. Resolving the orientation angles and determining the accurate rotational modes of the gold nanorod are critical in biological observations because the chirality of biological macromolecules and their assemblies, for example right-or left-handed helices, is fundamental in biology. Herein, we overcome this limitation by combining differential interference contrast (DIC) microscopy image pattern recognition with DIC polarization anisotropy analysis to resolve the exact azimuthal angles (from 08 to 3608) as well as the polar angles of tilted AuNRs that are positioned in the focal plane of the objective lens without sacrificing the spatial and temporal resolution. The rotational direction of individual in-focus AuNRs can thus be tracked dynamically. Finally, we successfully monitored the real-time rotational behavior of transferrin-modified gold nanorods on live cell membranes. Many biological processes involve rotational motion at the nanoscale, for example RNA folding,[2] walking of molecular motors,[3] twisting of dynamin assembly,[4] and self-rotation of ATPase.[5] Tracking the rotational motion with optical probes is of great importance to understanding these processes in biological and engineered environments. Fluorescence anisotropy has been commonly attempted to probe the rotational motion of biomolecules using organic dyes, conjugated polymers, and inorganic semiconductor nanocrystals.[6] Nevertheless, the major disadvantages of current fluorescent orientation probes are stochastic transition between on and off states,[7] high photobleaching tendency,[7a, 8] and less-than-desirable biocompatibility,[9] thus limiting their use in biological systems. Recently, AuNRs have gained considerable attention as suitable orientation probes because of their shape-induced anisotropic optical properties,[1a–c] large scattering and absorption cross-sections resulting from the surface plasmon resonance (SPR) effect, high chemical and photostability, and excellent biocompatibility.[10] Scattering-and absorptionbased polarization anisotropy measurements of AuNRs have been carried out under dark-field (DF) microscopy [1a] and photothermal heterodyne imaging.[1d] These methods were successfully used to measure the orientation of AuNRs. However, in these methods, only the in-plane orientation is effectively obtained while the out-of-plane orientation is still ambiguous. Furthermore, their applicability for studies of fast dynamics in live cells is limited. It is a challenge for DF microscopy to differentiate AuNRs from other highly scattering cellular components. Photothermal heterodyne imaging requires rapid scanning of the sample to collect an image and comprehensive intensity and polarization modulation of the heating beam.

DIC microscopy is better suited to probe orientation and rotational motion of nanoobjects in live cells when used in combination with plasmonic AuNRs.[1e] DIC microscopy resolves the optical path difference between two mutually orthogonally polarized beams separated by a shear distance along the optical axis of a Nomarski prism (Supporting Information, Figure S1). The nature of the interference makes it insensitive to the scattered light from surrounding cellular components and keeps its high-throughput capability. Therefore, the DIC microscopy-based single particle orientation and rotational …