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Cite this: DOI: 10.1039/c5nr03847b Received 11th June 2015, Accepted 2nd July 2015

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Characteristic image patterns of single anisotropic plasmonic nanoparticles embedded in a gel matrix† Ji Won Ha*

DOI: 10.1039/c5nr03847b www.rsc.org/nanoscale

We present characteristic doughnut-shaped image patterns of gold nanorods embedded in a thin layer of a gel matrix observed under a dark-field microscope. The characteristic scattering field distributions allow us to estimate the spatial orientation of single gold nanorods. The measured scattering patterns are further verified by a simulation study.

Rotational motion at the nanoscale is involved in many biological processes such as RNA folding,1 myosin walking,2 twisting of dynamin assembly,3 and self-rotation of ATPase.4 In this regard, single particle rotational tracking with optical probes is of great importance in elucidating these important processes in biological environments. Recently, gold nanorods (AuNRs) have been widely used as ideal probes for orientation sensing because of their shape-induced anisotropic optical properties,5,6 large scattering and absorption cross-sections resulting from the surface plasmon resonance (SPR) effect,7 high chemical and photostability, and excellent biocompatibility.8 A few polarization-based optical techniques including darkfield (DF) polarization microscopy,5 photothermal heterodyne imaging (PHI),9 differential interference contrast (DIC) polarization anisotropy,10,11 and total internal reflection (TIR) scattering microscopy12 have been used to determine the orientation of a AuNR. However, these polarization-based methods are limited by the angular degeneracy which refers to the inability to differentiate the nanorod orientations in the four quadrants of the Cartesian coordinate system due to their symmetric cylindrical shape. Recently, defocused orientation and position imaging (DOPI) techniques have been used to overcome the angular degeneracy in the polarization-based optical methods.2 DOPI techniques are more direct, simpler and easier than the polarization-based methods, and they have the capability of deter-

Department of Chemistry, University of Ulsan, 93 Daehak-Ro, Nam-Gu, Ulsan 680749, Republic of Korea. E-mail: [email protected]; Tel: +82 52 259 2347; Fax: +82 52 259 2348 † Electronic supplementary information (ESI) available: Additional experimental details and results. See DOI: 10.1039/c5nr03847b

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mining three-dimensional (3D) orientation of single AuNRs without the angular degeneracy.2,13–16 The core idea is that the direct detection of the spatial distribution of the scattered or emitted field of single dipoles becomes possible when the imaging system is deliberately defocused by ∼1 μm. However, it is still challenging to determine more accurate and reliable 3D orientation of a AuNR due to the deteriorated images with the largely reduced signal intensity in the DOPI techniques.2 To overcome the limitation of the DOPI methods, we recently reported total internal reflection (TIR) scatteringbased focused orientation and position imaging (FOPI) of AuNRs supported on a gold film.17 The core idea of the FOPI method is to use the strong coupling between plasmonic nanoparticles and dielectric supporting substrates.18–20 The characteristic scattering patterns of AuNRs on the gold substrate enabled high-throughput determination of the 3D spatial orientation of in-focus AuNRs within a single frame without angular degeneracy. Despite the advantages of the FOPI technique in single particle rotational tracking, the major limitation was the necessity of a strong interaction of AuNRs with a dielectric gold film. Therefore, it is important to achieve the characteristic doughnut-shaped scattering patterns without such an interaction. In the present study, we measured single AuNRs embedded in a gel matrix to better understand their characteristic scattering field distributions under a DF microscope. We experimentally reveal that the doughnut-shaped image patterns can be observed when a AuNR is aligned perpendicular to the glass surface without any interaction and field enhancement, which is further supported by a simulation study. We further demonstrate that the characteristic scattering patterns enable us to estimate the 3D spatial orientation of a AuNR. The AuNRs used in this study were purchased from Nanopartz (Loveland, CO, USA). Fig. S1A (ESI†) shows a scanning electron microscopy (SEM) image of the AuNRs, while Fig. S1B (ESI†) shows a UV-Vis absorption spectrum of the AuNRs dispersed in water. The transverse SPR peak appeared at 516 nm, while the longitudinal SPR peak appeared at 700 nm. The average length and width of AuNR were 73.7(±3.38) nm and

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25.4(±1.65) nm, respectively (Fig. S2, ESI†). The sample was prepared by spin casting AuNRs in 0.5% agarose gel on a precleaned glass slide, and the AuNRs were embedded in a thin layer of the gel matrix. The AuNRs were then measured by illuminating with randomly-polarized white light tightly focused by a high numerical aperture (NA) oil condenser under a DF microscope. The gel matrix allowed the AuNRs to be fixed with a variety of 3D orientations, enabling us to study their characteristic orientation-dependent scattering patterns by DF microscopy. According to the electrostatic approximation, plasmon oscillations from anisotropic AuNRs can be simplified as three-perpendicular independent dipoles along the three axes (Fig. 1A); oscillation along the long principal axis (a-axis) is defined as a longitudinal mode and the other perpendicular oscillations are defined as transverse modes vibrating along the short axes (b and c axes). Ea indicates the scattering electric field of the nanorod along the main long axis, while Eb and Ec are the scattering electric field along the short transverse axes b and c (Fig. 1A). In this study, the polar angle θ and the azimuthal angle φ of a AuNR in 3D space are defined in Fig. 1A. AuNRs that are much smaller than the wavelength of the incident light can be considered electric point dipoles. The overall scattering electrical field from a AuNR can be quantified through linear superposition of three independent scattering

Fig. 1 Scattering patterns of AuNRs in a gel matrix under randomlypolarized white light illumination. (A) Schematic depicting three-perpendicular dipoles along the three axes. Ea denotes the scattering electric field of the nanorod along the main long axis. Definitions of the polar angle θ and the azimuthal angle φ of a AuNR in 3D space are also shown. (B) DF scattering image of AuNRs with different image patterns (a doughnut-shaped pattern vs. a solid-bright spot). (C) Schematic of AuNRs in a gel matrix to produce a doughnut-shaped scattering pattern (left) and a solid bright spot (right).

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electric fields associated with three mutually orthogonal dipoles as shown in eqn (1):13 X EðscatÞ ¼ EðscatÞ j ¼ EðscatÞa þ EðscatÞb þ EðscatÞc ð1Þ a;b;c

In the previous study, we reported the FOPI method to resolve the 3D spatial distribution of the scattered electric field arising from the three mutually orthogonal oscillation dipoles of a AuNR at the focal plane without suffering from the greatly reduced signal intensity.17 However, the major limitation of the FOPI method was the necessity of the strong coupling (or constructive interference) of a AuNR’s out-of-plane transverse dipole with an image charge dipole created in a gold substrate. In the present study, we tried to achieve the doughnutshaped scattering patterns of a AuNR at the focal plane without such an interaction and electric field enhancement. The idea comes from the fact that the scattering electric field from the longitudinal axis (Ea) of a AuNR is much more dominant than the combined electric field from the transverse axes (Eb + Ec) under a DF microscope with randomly-polarized white light illumination. We therefore expect that the doughnut-shaped scattering pattern could be observed for a AuNR standing upright (θ = 0°) on a glass slide without any field enhancement at the focal plane because of the strong electric field from the longitudinal axis. First, we experimentally checked if the doughnut-shaped scattering patterns can be observed for AuNRs standing upright on a glass slide without any interaction with the surrounding environment. In this study, we embedded AuNRs in a gel matrix because they can have random polar angles (θ) between 0° and 90°. We then measured the embedded AuNRs by scanning in the z-direction with a vertical step size of ∼40 nm. Fig. S3 (ESI†) shows a DF scattering image of many AuNRs in the gel matrix. The vertical scan allowed us to obtain both focused and defocused images of randomly-orientated AuNRs fixed in the gel matrix. As shown in Fig. 1B, doughnutshaped scattering patterns are observed for some AuNRs while solid bright spots are observed for other AuNRs. The different image patterns can be ascribed to the different orientations of the dipole along the long axis of a AuNR (Fig. 1C). More specifically, if the dipole from the longitudinal axis of a AuNR is orientated perpendicular to the glass substrate, a doughnutshaped scattering pattern is detected by the objective lens.18 However, if the dipole from the longitudinal axis is orientated parallel to the substrate, a solid bright spot is detected by the objective lens (Fig. 1C).18 Therefore, we confirmed that the doughnut-shaped scattering patterns can also be detected for a AuNR orientated perpendicular to the glass substrate. To the best of our knowledge, this is the first report to show the doughnut-shaped scattering patterns of in-focus AuNRs without any further interaction with the dielectric substrates under DF microscopy. Next, we tried to take a closer look at the doughnut-shaped scattering patterns that can provide valuable information on AuNRs. It is notable that the doughnut-shaped scattering patterns are different in the intensity at the center (Fig. 1B).

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To further clarify this, we chose four single AuNRs showing doughnut-shaped scattering patterns with different intensities at the center in the focal plane, and the intensity line sections were drawn across the center of the scattering patterns (Fig. 2). The difference in the intensity at the center could be mainly attributed to the different degree of contribution of the scattering electric field from the transverse axes,18 which is directly related to the width of a AuNR. To verify this, we further simulated doughnut-shaped scattering patterns of a AuNR by varying the degree of contribution from the transverse axes. For this purpose, we used the simulation program developed by Enderlein and Böhmer for calculating the characteristic intensity distribution from an emitter with three perpendicular emission dipoles of different emission strength.14 In this simulation, there are two important parameters of κ and R which allow us to define the emission strength ratios of the three independent dipoles. The ratio R defines the emission strength of the a-dipole (or longitudinal dipole) to the combined b and c transverse dipoles as shown in eqn (2): R  I a þ ð1  RÞ ðI b þ I c Þ

ð2Þ

When R is 1, we only have the contribution from a-dipole (longitudinal dipole) to the image patterns. This can be the

Fig. 2 Doughnut-shaped scattering patterns with a different intensity at the center. (A) Dependence of the R value on the doughnut-shaped scattering pattern of a AuNR. With decreasing R value, the intensity at the center increased because of the increased contribution from the transverse dipoles. The simulated scattering patterns matched well with the measured patterns. (B) The intensity line sections across the center of the four doughnut-shaped scattering patterns in (A). It is clearly shown that the intensity at the center increases from NR 1 to NR 4.

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case of single fluorescent molecules with a single principal dipole. However, in the case of AuNR the other two transverse dipoles (b and c) can contribute to the image patterns. In the simulation, we can increase the degree of contribution from the transverse dipoles by decreasing the ratio R value (e.g., from 1 to 0.8). Fig. S4 (ESI†) shows the simulated scattering patterns of a AuNR by varying the parameter R from 1 to 0. In this simulation, the polar angle θ of a AuNR was set to 0°. There are important observations that need to be discussed as shown in Fig. S4 (ESI†). It is found that a doughnut-shaped scattering pattern changes to a solid bright spot as the R value decreases from 1 to 0. It is notable that the doughnut-shaped scattering pattern is clearly observed when R is greater than 0.8 while the intensity at the center of the doughnut pattern becomes higher with decreasing R from 1 to 0.8. Furthermore, the size of the simulated pattern becomes smaller when the R value decreases. The results indicate that the thickness of single AuNRs can be estimated from their characteristic scattering patterns with respect to shape, size, and the intensity at the center. We then tried to confirm if we can directly estimate the 3D orientation of AuNRs in the gel matrix from their characteristic spatial scattering intensity distributions. We first simulated scattering patterns by changing the polar angle θ of a AuNR from 0° to 90° at the fixed ratio R of 1 in the focal plane (Fig. S5, ESI†). A doughnut-shaped scattering pattern at the polar angle of 0° is circularly symmetric. However, a dipole torus is no longer circularly symmetric and the center moves toward the edge of a pattern with increasing the polar angle θ. In addition, doughnut-shaped scattering patterns are clearly observed when the polar angle θ is less than 40° in case R = 1. At the polar angle greater than 40° a solid bright spot elongated along the main long axis of a AuNR appears. This is because the strength of longitudinal dipole oscillation significantly dominates the transverse dipole oscillations for elongated AuNRs. Please note that this elongated spot is also distinguishable from the circularly symmetric solid bright spot shown in Fig. S4 (when θ = 0°, ESI†). We further simulated scattering patterns of a AuNR at the different R values of 0.85 and 0.75 (Fig. S6 and S7, ESI†), and the same trend was observed in the change of scattering patterns. Therefore, we found that the characteristic scattering patterns can enable us to estimate the 3D spatial orientation of a AuNR. To experimentally verify the capability of estimating the spatial orientation of a AuNR, three single AuNRs with different orientation angles in the Cartesian plane were randomly selected (Fig. 3). The measured scattering patterns at the focal plane were used to estimate the spatial orientation of the AuNRs by fitting with the best-matched simulated patterns. We found that the measured patterns for the three AuNRs showed a good agreement with the best-matched simulation patterns (Fig. 3). It should be noted that the azimuthal angles are resolved without angular degeneracy in the four quadrants of the Cartesian plane. Despite the capability of resolving the orientation angles of AuNRs at the focal plane, it

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Fig. 4 Correlated DF (A) and DIC (B) images for same AuNRs. (C, D) Enlarged DF and DIC images for two AuNRs with different scattering patterns of solid spot (C) and doughnut (D). Fig. 3 The measured and best-matched simulation patterns from three randomly selected AuNRs in different orientations inside gel matrix. The measured patterns (A–C) are obtained at the focal plane. It is shown that the measured patterns (A–C) match well with the simulated patterns (D–F). The 3D spatial orientations of the three AuNRs determined through the pattern match analysis are illustrated (G–I). The scale bar represents 1 μm.

is still challenging for this method to estimate the spatial orientations of in-focus AuNRs with large polar angles (e.g., θ = 90°, a nanorod lying flat to the surface). The limitation, however, can be overcome by combining with a defocused orientation imaging technique (Fig S8, ESI†). To gain further insight into the characteristic image patterns of AuNRs, we performed a correlation study between DF scattering images and DIC interference images for the same AuNRs. We first measured AuNRs with randomly polarized white light under DF microscopy, and then we carried out DIC imaging for the same AuNRs under 700 nm excitation close to the longitudinal SPR of the AuNRs in water (Fig. S1, ESI†). From the correlated DF and DIC images (Fig. 4) we found that solid bright spots in the DF image show a typical DIC image of in-plane AuNR with two bright and dark portions (Fig. 4C). More notably, we found that doughnut-shaped DF scattering patterns of AuNRs standing upright show a crossed DIC image pattern with the four bright and dark portions (Fig. 4D). This is consistent with our previous report for DIC images of a tilted AuNR.21 To the best of our knowledge, this is the first report to show the characteristic scattering images correlated with the characteristic interference images for the same AuNRs. Therefore, the results provide a better understanding of the scattering and interference image patterns of AuNRs with different orientations under DF and DIC microscopes. Furthermore, the correlation study supports that the doughnut-shaped scattering patterns result from tilted and vertical AuNRs. Lastly, we checked if we can observe and track the change of doughnut-shaped scattering patterns of a AuNR in dynamic

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biological systems as a function of time. In this study, we chose single AuNRs rotating on synthetic membranes as a model system. Cetyltrimethyl ammonium bromide (CTAB)coated AuNRs were introduced into a chamber. The initially freely diffusing AuNRs were bound to the membrane through non-specific interactions. We recorded movies that show rotational motions of surface-bound AuNRs at the temporal resolution of 50 ms under DF microscopy. Fig. 5A shows a DF image of AuNRs bound onto the membrane. We can observe

Fig. 5 Direct observation of the orientation of AuNRs rotating on synthetic membrane. (A) DF scattering image of AuNRs bound onto the synthetic membrane. A doughnut-shaped scattering pattern is observed for the AuNR 10 squared with white. (B) Schematic to depict the conformation of the AuNR 10 on the membrane. The doughnut shape indicates that the AuNR is standing up on the membrane as shown in (B). (C) 10successive DF images of the AuNR 10 as a function of time. The temporal resolution is 50 ms. The white arrows show the estimated in-plane orientation of the AuNR 10. It is found that the doughnut-shaped patterns are changed as a function of time. (D) Polar graph to present the azimuthal angle of the AuNR 10 as a function time for the image sequence of (C). The azimuthal angle was estimated from the doughnut-shaped scattering patterns over time.

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that the AuNR 10 bound to the membrane showed the doughnut-shaped scattering pattern (Fig. 5A). The doughnut shape indicates that the AuNR 10 is standing upright on the membrane as depicted in Fig. 5B. We chose 10-consecutive frames from a movie (Fig. 5C). We observed that the doughnut-shaped patterns change dynamically as a function of time (Fig. 5C). Fig. 5D is a polar graph to show the rotational track of the in-plane orientation angle φ as a function of time for the image sequences. The orientation angle φ for the image frames was estimated from the characteristic scattering patterns. Furthermore, the parameter R value for the AuNR 10 was estimated to be ∼0.9, and the polar angle θ was changed between 0° and 35° for the 10-image sequences. Another example of a AuNR rotating on the membrane is provided in Fig. S9 (ESI†). Therefore, we could observe the change of the doughnut-shaped scattering pattern of a AuNR bound onto the membrane as a function of time. In conclusion, we present a characteristic doughnut-shaped image pattern of single AuNRs embedded in a gel matrix. This method does not require a strong interaction (or coupling) between a AuNR and a dielectric substrate. We demonstrated that the characteristic doughnut-shaped image patterns can be used to estimate the 3D spatial orientation of a AuNR at the focal plane. Therefore, the results provide a deeper understanding of the characteristic image patterns of AuNRs under DF and DIC microscopes.

Acknowledgements 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 Laboratory is operated for the U.S. Department of Energy by Iowa State University under contract no. DE-AC0207CH11358. This work was also supported by the University of Ulsan, South Korea.

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2 E. Toprak, J. Enderlein, S. Syed, S. A. McKinney, R. G. Petschek, T. Ha, Y. E. Goldman and P. R. Selvin, Proc. Natl. Acad. Sci. U. S. A., 2006, 103, 6495–6499. 3 A. Roux, K. Uyhazi, A. Frost and P. De Camilli, Nature, 2006, 441, 528–531. 4 T. Nishizaka, K. Oiwa, H. Noji, S. Kimura, E. Muneyuki, M. Yoshida and K. Kinosita, Nat. Struct. Mol. Biol., 2004, 11, 142–148. 5 C. Sönnichsen and A. P. Alivisatos, Nano Lett., 2004, 5, 301– 304. 6 L. Xiao, Y. Qiao, Y. He and E. S. Yeung, J. Am. Chem. Soc., 2011, 133, 10638–10645. 7 S. Link and M. A. El-Sayed, J. Phys. Chem. B, 1999, 103, 8410–8426. 8 C. J. Murphy, A. M. Gole, J. W. Stone, P. N. Sisco, A. M. Alkilany, E. C. Goldsmith and S. C. Baxter, Acc. Chem. Res., 2008, 41, 1721–1730. 9 W.-S. Chang, J. W. Ha, L. S. Slaughter and S. Link, Proc. Natl. Acad. Sci. U. S. A., 2010, 107, 2781–2786. 10 J. W. Ha, W. Sun, G. Wang and N. Fang, Chem. Commun., 2011, 47, 7743–7745. 11 J. W. Ha, W. Sun, A. S. Stender and N. Fang, J. Phys. Chem. C, 2012, 116, 2766–2771. 12 K. Marchuk and N. Fang, Nano Lett., 2013, 13, 5414–5419. 13 L. Xiao, Y. Qiao, Y. He and E. S. Yeung, Anal. Chem., 2010, 82, 5268–5274. 14 M. Böhmer and J. Enderlein, J. Opt. Soc. Am. B, 2003, 20, 554–559. 15 M. A. Lieb, J. M. Zavislan and L. Novotny, J. Opt. Soc. Am. B, 2004, 21, 1210–1215. 16 T. Li, Q. Li, Y. Xu, X.-J. Chen, Q.-F. Dai, H. Liu, S. Lan, S. Tie and L.-J. Wu, ACS Nano, 2012, 6, 1268–1277. 17 J. W. Ha, K. Marchuk and N. Fang, Nano Lett., 2012, 12, 4282–4288. 18 H. Chen, T. Ming, S. Zhang, Z. Jin, B. Yang and J. Wang, ACS Nano, 2011, 5, 4865–4877. 19 J. J. Mock, R. T. Hill, A. Degiron, S. Zauscher, A. Chilkoti and D. R. Smith, Nano Lett., 2008, 8, 2245–2252. 20 P. Swanglap, L. S. Slaughter, W.-S. Chang, B. Willingham, B. P. Khanal, E. R. Zubarev and S. Link, ACS Nano, 2011, 5, 4892–4901. 21 L. Xiao, J. W. Ha, L. Wei, G. Wang and N. Fang, Angew. Chem., Int. Ed., 2012, 51, 7734–7738.

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Characteristic image patterns of single anisotropic plasmonic nanoparticles embedded in a gel matrix.

We present characteristic doughnut-shaped image patterns of gold nanorods embedded in a thin layer of a gel matrix observed under a dark-field microsc...
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