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SERS Orientational Imaging of Silver Nanoparticle Dimers Sarah M. Stranahan, Eric J. Titus, and Katherine A. Willets* Department of Chemistry and Biochemistry, The University of Texas at Austin, 1 University Station A5300, Austin, Texas 78712, United States

bS Supporting Information ABSTRACT: This Article introduces surface-enhanced Raman scattering (SERS) orientational imaging as a powerful far-field optical technique for determining the in-plane and out-of-plane orientations of SERSactive nanoparticle dimers. Optical images of Rhodamine 6G (R6G) SERS emission patterns are measured and correlated with atomic force microscopy (AFM) images of the associated SERS-active silver nanoparticle dimers. The AFM is used to measure individual silver nanoparticle dimer orientations and height asymmetry, defining in-plane and out-of-plane angles associated with the dimer geometry. Theoretical emission pattern images based on these angles are generated using a simple dipole emission model and show excellent agreement with the experimental emission patterns. This technique provides a rapid all-optical technique to analyze the orientation of SERS active nanoparticle dimers. SECTION: Nanoparticles and Nanostructures

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oble metal nanoparticle dimers are used as substrates in surface-enhanced spectroscopies, enabling highly sensitive molecular detection.1 8 The polarization-dependent optical properties of these aggregates are dictated by their orientation relative to the sample plane.9,10 Dimer orientation has conventionally been determined through structural characterization techniques, such as atomic force microscopy (AFM) and electron microscopy, or through optical anisotropy measurements, in which the scattering or emission intensity originating from the nanoparticle dimer is recorded as a function of a rotating polarization analyzer.9,11 15 These techniques are time-consuming and are not readily able to provide out-of-plane orientations in the case of a vertically oriented nanoparticle dimer. Here we introduce surface-enhanced Raman scattering (SERS) orientational imaging as an all-optical, far-field imaging technique able to determine silver nanoparticle dimer orientations as well as nanoparticle height asymmetry quickly in a single experiment. Orientational imaging has long been used in the singlemolecule fluorescence community to determine the orientation of single fluorophores and quantum dot emission and has recently been applied to Rayleigh scattering from a single gold nanorod.16 23 In this far-field imaging technique, angular lobes appear in the diffraction-limited image of a single emitter, which correspond to the orientation of the emission dipole (in the case of a single fluorophore) or the longitudinal dipole axis of the scattering nanorod. Often, these images are obtained by slightly defocusing the optical image to enhance the contrast in the lobes of the dipole emission pattern; this is due to the low signal-tonoise typically associated with these emitters (single-molecule fluorophores in particular). Here we are able to image the angular lobes while the sample is in focus because of the high signal-tonoise associated with the strong bursts of SERS activity. For our studies, we describe SERS emission from a nanoparticle dimer as a simple dipole emitter, using the angles as r 2011 American Chemical Society

described in Figure 1A. The first angle (j) defines the orientation of the long axis of the nanoparticle dimer in the sample plane (Figure 1B), and the second angle (θ) defines the tilt (out-ofplane) angle of the dimer from the optical axis, defined by the relative height of the two nanoparticles (Figure 1C). Although modeling a nanoparticle dimer as a dipole is a simple approximation, previous work in the literature suggests that this is a valid strategy for modeling our data.9,14 For example, Haran et al. have used polarization anisotropy measurements to show that singlemolecule SERS (SM-SERS) from a nanoparticle dimer behaves as a single dipole oriented parallel to the long axis of the two nanoparticles, whereas K€all et al. have used Fourier plane imaging of nanoparticle dimers decorated with ∼10 000 molecules to demonstrate similar dipole-like behavior.9,14 These data, along with our own data shown below, support a simple dipole as a valid model for SERS emission from a nanoparticle dimer. Figure 2 shows three examples of Rhodamine 6G (R6G) SERS emission patterns (center column) and the associated nanoparticle dimer structures, as determined by AFM (left column). Each emission image corresponds to frames exhibiting strong intensity bursts in our image stack (Figure S-1 of the Supporting Information), which is consistent with R6G SERS activity (Figure S-2 of the Supporting Information).8,15 To obtain SERS emission patterns, such as those shown in Figure 2, we sum together several image frames (typically, 3 6) that correspond to strong intensity bursts. Although summing multiple image frames is not necessary for observing the lobes associated with directional SERS emission in these samples at our 0.1 s integration time (Figure S-11 of the Supporting Information), adding the frames together provides higher contrast and allows for more Received: August 19, 2011 Accepted: October 10, 2011 Published: October 10, 2011 2711

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Figure 2. (A,D,G) AFM topography images of three SERS-active silver nanoparticle dimers with (B,E,H) correlated optical images of R6G SERS emission patterns originating from the respective dimers and (C,F,I) theoretical dipole emission patterns. The angles for each are as follows: (A C) j = 60°, θ = 90°; (D F) j = 115°, θ = 84°; and (G I) j = 100°, θ = 78°. The AFM image scale bar is 100 nm, and the intensity scale bar is in units of nanometers. The optical image scale bar is 500 nm. Emission patterns in panels B and E are summed over four image frames, whereas panel H is summed over three image frames.

Figure 1. (A) Single emitting dipole (red arrow) can be defined by two angles in a 3D coordinate system where phi (j) defines the rotation of the dipole in the sample plane (x y plane) in reference to the x axis and theta (θ) defines the tilt of the dipole in reference to the optical axis (z). (B) Three examples of dimers with varied phi values and (C) three examples of dimers with varied theta values. For the asymmetric nanoparticle pair in panel C, the value of theta is calculated using the center-to-center distance, as shown by the red arrow.

straightforward comparisons with theory. Image frames corresponding to weak background emission, typically assigned to nanoparticle luminescence, do not show emission patterns with the 0.1 s of integration time used in these studies (Figure S-11 of the Supporting Information). Using a Matlab interface called QDControl, developed and made available online by J€ org Enderlein, we are able to produce an exact wave-optical calculation of an image of a single emitting dipole at an air glass interface (Figure 2, right column).23 The required input consists of two angles describing the dipole orientation (j and θ), the image magnification, the N.A. of the objective, and the amount of image defocusing. (Several other

parameters are available to account for multiple dipole axes, but these are set to include only contributions from a single dipole.) We set the amount of image defocusing in our calculations to 0 nm, which shows strong agreement between our experimental and theoretical results. Because this program calculates emission patterns at an air glass interface, we have used a glass substrate in our experiments for direct comparison with theory; therefore, we use AFM for structural characterization of our samples because it is compatible with this substrate, unlike electron microscopy. (See Figure S-9 of the Supporting Information.) The resolution of our AFM is sufficient to confirm that we are imaging nanoparticle dimers and also provides the orientation of the nanoparticle junction used to determine j and (unlike electron microscopy) the critical height information that we require for our calculation of the out-of-plane dipole angle, θ. Figure 2A shows a dimer of nanoparticles with equal heights based on the AFM topography and the dimer long-axis rotated ∼60° (j) from the x axis. We expect the plasmon mode enabling SERS to lie parallel to the long axis of the dimer (j = 60°) and in the plane of the sample (θ = 90°) due to the equal heights of the two nanoparticles. (See Figures S-5 to S-8 of the Supporting Information for AFM cross sections of all nanoparticles discussed herein.) Figure 2B shows the experimental SERS emission image corresponding to the nanoparticle dimer in Figure 2A, and Figure 2C shows the calculated emission pattern based on the angles determined from the AFM image. The theoretical emission image is in excellent agreement with the experimental image, 2712

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Figure 3. R6G SERS emission patterns (A C) originating from the silver nanoparticle in (D) upon excitation with light polarized at 90, 45, and 0°. (E) Theoretical dipole emission pattern using j = 75 and 96°. Five images are summed to produce the optical images in (A) and (B), whereas 10 images are summed to produce the image shown in panel C. The AFM image scale bar is 100 nm, and the optical image scale bar is 500 nm.

confirming that SERS emission originating from a nanoparticle dimer can be modeled as a single emitting dipole described by the structure and orientation of the dimer relative to the substrate. The second nanoparticle dimer example (Figure 2D) is made of nanoparticles with unequal heights, resulting in a tilt angle (θ) of ∼84° from the optical axis based on the nanoparticle heights. Again, we expect the in-plane component of the emission to be oriented parallel to the long axis of the nanoparticle dimer (j = 115°). Using these values of j and θ, we obtain the theoretical emission image in Figure 2F, which is in near perfect agreement with the measured emission pattern in Figure 2E. The third dimer example (Figure 2G) consists of a nanoparticle dimer with a more extreme height and size difference between the nanoparticles based on our AFM measurements. The dipole tilt (θ) associated with this asymmetric dimer is ∼78° from the optical axis based on our height measurements. The associated SERS emission pattern (Figure 2H) shows a small degree of asymmetry in the emission pattern lobes, with the ends of the lobes appearing blunted at the lower right of the image and appearing sharper and extending further past the central feature at the upper left of the image. A similar trend is observed in the emission pattern lobes in the theoretical image (Figure 2I), but there is a notable elongation observed in the central feature in the calculated emission pattern, which is not observed in the experimental image. This suggests that the dipole model may not be appropriate for highly asymmetric systems and that electrodynamic calculations may be required to model the emission behavior of more complex systems fully. Nonetheless, even with our simplified dipole model, we still find strong agreement between our experimental and calculated images and can optically determine the orientation of the SERS-active dimer based on the alignment of the emission pattern lobes. One possible concern with these data is how much of the signal is contributed by photons from R6G SERS versus photons due to the well-known continuum background that typically accompanies SM-SERS experiments or even stray R6G fluorescence.13,24,25 Brus and coworkers have noted that the

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Figure 4. Experimental SERS emission patterns (A,B) and corresponding theoretical dipole emission patterns (C: j = 55°; θ = 34°) and (D: j = 0°; θ = 0°) arising from silver nanoparticle aggregates with strong out-of-plane plasmon modes. A is summed over six image frames, whereas B is summed over five image frames. The scale bar is 600 nm.

continuum background can dominate SERS spectra of R6G on silver colloids and have proposed that electron tunneling between the SERS analyte and the silver nanoparticle is responsible for this background.26 Stray R6G fluorescence may also contribute photons to the overall signal, although we expect fluorescence to be strongly quenched on the silver surface and to couple to the radiative plasmon mode in a similar manner to the SERS signal. In our experiments, we note that the photons contributed from non-SERS sources can dominate by up to a factor of 10, yet it is impossible to separate the sources of background from our SERS signal because the two are intimately connected and one is not observed without the other.13 This confirms that the emission patterns are a SERS-mediated phenomenon. We also note that images collected from a different dye, Nile Blue, which shows a much smaller background than R6G and has no fluorescence contribution, still result in strong emission patterns (Figure S-3 of the Supporting Information). We now look closer at the polarization behavior of our SERS emission patterns. We imaged R6G SERS originating from the nanoparticle dimer in Figure 3D, while rotating the excitation polarization to 90, 45, and 0° (Figure 3A C). The calculated emission pattern based on the AFM image is shown in Figure 3E. The lobes of the SERS dipole emission pattern are oriented perpendicular to the long axis of the nanoparticle dimer, as before, and their location remains unchanged at each excitation polarization, as expected. The intensity of the SERS does change in response to the excitation polarization rotation, with the highest intensity occurring when the excitation polarization is parallel to the long-axis of the nanoparticle dimer. If the dipole model was insufficient to describe our data, we would expect the emission pattern to change in response to different excitation polarizations, due to excitations of different plasmon modes. However, given that our data are well-modeled as a dipole, independent of excitation polarization, we are confident that this is a reasonable model to describe this nanoparticle dimer system. All of the examples we have discussed up to this point are made of two nanoparticles with the long axis oriented roughly in the sample plane, with at most a 12° tilt from the substrate. We now look at SERS emission patterns consistent with strong 2713

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The Journal of Physical Chemistry Letters out-of-plane dipole components in Figure 4. Corresponding AFM images (Figures S-6 to S-8 of the Supporting Information) provide no insight into the geometry of the nanoparticle aggregates in these examples; this is because AFM is only sensitive to height and cannot report on how many objects contribute to the measured height in the case of stacked nanoparticles. However, the SERS emission patterns shown in Figure 4 tell us that the nanoparticles must have a strong out-of-plane component due to the “donut”-like emission patterns. For example, we can match the experimentally observed SERS emission pattern in Figure 4A to a theoretical prediction (Figure 4C) in which the dipolar SERS active plasmon mode is tilted 34° from the optical axis and is rotated 55° from the x axis. In Figure 4B, we observe a nearly radially symmetric donut pattern, indicative of a radiating dipole oriented almost perfectly perpendicular to the sample plane (θ = 0°). Although the image in Figure 4B shows a slight intensity bias toward the upper left, we have modeled this as a vertically oriented dipole due to subsequent images of the same region of interest that do not show this bias (Figure S-11 of the Supporting Information). In the case of the corresponding theoretical map (Figure 4D), we arbitrarily choose j = 0°, although symmetry will produce the same image, regardless of the value of j. The data shown in Figures 2 4 all highlight our ability to determine the orientation of a SERS-active nanoparticle dimer simply by observing the SERS emission image acquired from that dimer. All three figures show that SERS emission pattern imaging is able to determine clearly the rotation of a nanoparticle dimer in the sample plane, and Figure 2G demonstrates that we are sensitive to at least a 12° tilt of the SERS active plasmon mode away from the sample plane, resulting from differences in nanoparticle heights. Moreover, the use of a high numerical aperture microscope objective introduces a sufficient z-polarized component to our excitation light such that we are able to excite, and observe, out-of-plane emission patterns using standard wide-field epiillumination.16,27 Although the overall intensity of these modes is reduced compared with the other examples (Figure S-1 of the Supporting Information), we are able to resolve the emission patterns clearly at our 0.1 s integration time (Figure S-11 of the Supporting Information). SERS emission pattern imaging provides a convenient alloptical technique able to identify nanoparticle dimer orientation that does not require transferring the nanoparticle sample to a secondary instrument (like an AFM or SEM) and does not require additional components in the optical setup (like anisotropy or Fourier plane imaging). We have verified this claim in Figure 4 by showing examples of SERS emission patterns where we can easily determine the emission orientation, despite the inadequate structural information provided by the AFM. We have observed that slightly defocusing the SERS emission image exaggerates the resulting pattern, as expected (Figure S-13 of the Supporting Information), and we will perform a systematic defocusing study in the future with the addition of a z-axis piezo stepper to our system. We have also observed that higher order nanoparticle aggregates (trimers, tetramers, etc.) yield strong SERS emission patterns, but these examples require a more rigorous theoretical analysis and will be the subject of a future publication. In conclusion, we have introduced SERS orientational imaging as a simple technique able to determine the orientations of SERSactive silver nanoparticle dimers. We have shown SERS emission patterns can be modeled as single emission dipoles with excellent agreement between experimentally observed SERS emission

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patterns and theoretical single emitting dipole images modeled with parameters determined by our correlated AFM images. We used correlated AFM images as a proof-of-concept study to confirm that SERS emission patterns report on the SERS active plasmon modes of nanoparticle dimers. Importantly, SERS orientational imaging can be a stand-alone technique used to determine silver nanoparticle dimer orientations, which is especially important when the nanoparticle geometry leads to vertically oriented SERS active plasmon modes that are extremely challenging to identify with traditional structural characterization techniques. We believe this approach will have utility for rapid structural analysis of rationally designed nanoparticle dimers for surface-enhanced spectroscopy as well as potential for resolving dimeric structures in more complex aggregated systems.

’ EXPERIMENTAL METHODS We prepare SERS samples with R6G adsorbed on silver colloid aggregates prepared by the Lee and Meisel method.28 The R6G concentration (1 nM) is in the regime typically associated with single-molecule SERS.13,29 R6G SERS is excited by linear polarized 6 mW 532 nm laser in epi-illumination using a 100, NA 1.3 oil immersion microscope objective. The resulting SERS scatter is collected by the same objective and passed through appropriate dichroic and long-pass filters and imaged onto a Princeton Instruments ProEM 512 electron-multiplied chargecoupled device (EM-CCD) camera. Dark-field scattering images of the same region of interest are also collected using a dark-field condenser in a transmission geometry. After optical imaging, the sample is transferred to an AFM (NT-MDT, NTegra Vita) mounted on top of an optical microscope for structural imaging. The AFM also has simultaneous dark-field imaging capability for pattern matching between the optical and AFM images (Figure S-4 of the Supporting Information).30 Moreover, the sample is prepared on a glass slide patterned with an alpha-numeric alignment grid allowing rapid image registration between the optical setup and an AFM for optical-structural correlation (Figure S-4 of the Supporting Information). ’ ASSOCIATED CONTENT

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Supporting Information. Experimental details, intensity time traces, description of the alignment strategy, AFM cross sections, AFM images of vertically aligned nanoparticles, SEM images, and defocused images. This material is available free of charge via the Internet at http://pubs.acs.org.

’ AUTHOR INFORMATION Corresponding Author

*E-mail: [email protected].

’ ACKNOWLEDGMENT We thank Dr. Kathryn Mayer and Dr. Hugo Celio for assistance with the ESEM and Maggie Weber for SEM images (Supporting Information). S.M.S. gratefully acknowledges the NSF IGERT “Atomic and Molecular Imaging” Program (grant no. DGE-0549417) for fellowship support. We also thank J€org Enderlein for graciously providing his defocused imaging MatLab scripts online and the Welch Foundation in support of the facilities utilized in the Center for Nano and Molecular Science at the University of Texas at Austin. This material is 2714

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The Journal of Physical Chemistry Letters based on work supported by the Welch Foundation under award no. F-1699 and the Air Force Office of Scientific Research under AFOSR award no. FA9550-09-0112.

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