Nanoparticle Tracking DOI: 10.1002/anie.201202340 Determining the Full Three-Dimensional Orientation of Single Anisotropic Nanoparticles by Differential Interference Contrast Microscopy** Lehui Xiao, JiWon Ha, Lin Wei, Gufeng Wang, and Ning Fang* 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 in- focus 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 interfer- ence 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 success- fully monitored the real-time rotational behavior of trans- ferrin-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. Fluo- rescence anisotropy has been commonly attempted to probe the rotational motion of biomolecules using organic dyes, conjugated polymers, and inorganic semiconductor nano- crystals. [6] Nevertheless, the major disadvantages of current fluorescent orientation probes are stochastic transition between on and off states, [7] high photobleaching tenden- cy, [7a, 8] and less-than-desirable biocompatibility, [9] thus limit- ing 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 absorp- tion cross-sections resulting from the surface plasmon reso- nance (SPR) effect, high chemical and photostability, and excellent biocompatibility. [10] Scattering- and absorption- based 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 scatter- ing 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. There- fore, the DIC microscopy-based single particle orientation and rotational tracking (SPORT) technique is more appli- cable to the studies of fast rotational dynamics in live cells. When plasmonic nanoparticles are illuminated under an optical microscope, the incident electromagnetic wave is commonly attenuated through absorption and scattering. [11] The attenuation at a given wavelength is quantified by the corresponding cross-section. Typically, when a AuNR is excited by monochromatic light at the longitudinal SPR wavelength, the scattered electromagnetic field can be simplified as generated from a single dipole with the oscillation direction along its principal axis, provided that the nanorod is much smaller than the wavelength of light. [11] [*] Dr. L. Xiao, [+] J. W. Ha, [+] L. Wei, Dr. G. Wang, Prof. N. Fang Ames Laboratory, U.S. Department of Energy, and Department of Chemistry, Iowa State University Ames, IA 50011 (USA) E-mail: nfang@iastate.edu Dr. L. Xiao, [+] L. Wei Biomedical Engineering Center, State Key Laboratory of Chemo/ Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Hunan University Changsha, 410082 (P.R. China) [ + ] These authors contributed equally to this work. [**] This work was supported by the U.S. Department of Energy, Office of Basic Energy Sciences, Division of Chemical Sciences, Geosciences, and Biosciences through the Ames Laboratory. The Ames Labo- ratory is operated for the U.S. Department of Energy by Iowa State University under contract no. DE-AC02-07CH11358. L.X. expresses thanks for the partial support from the Scholarship Award for Excellent Doctoral Student from the Ministry of Education, China. Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/anie.201202340. . Angewandte Communications 7734  2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim Angew. Chem. Int. Ed. 2012, 51, 7734 –7738