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Correlated Super-Resolution Optical and Structural Studies of Surface-Enhanced Raman Scattering Hot Spots in Silver Colloid Aggregates Maggie L. Weber 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: In order to advance the field of single-molecule surface-enhanced Raman scattering (SM-SERS), a better understanding of colloid morphology and hot spot properties in noble metal nanoparticle aggregates is crucial. We present super-resolution optical studies of surface-enhanced Raman scattering (SERS) from rhodamine 6G (R6G) molecules adsorbed onto silver colloid aggregates correlated with scanning electron microscope (SEM) images of those same aggregates. The scattering intensity maps of the SERS signal, obtained from the super-resolution fits, are overlaid with SEM topographical images of the colloids to map the shape of the SERS hot spot and the spatial origin of SERS intensity fluctuations with sub-5 nm resolution. These results have vital implications for developing reproducible and robust substrates capable of SM-SERS. SECTION: Nanoparticles and Nanostructures

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ingle-molecule surface-enhanced Raman scattering (SM-SERS) enables detection of Raman vibrational modes from single molecules adsorbed onto noble metal nanoparticle aggregates.1
6 Theoretical calculations show that electromagnetic fields can be strongly enhanced in the nanoparticle aggregate junction regions, or “hot spots”, increasing the Raman scatter by many orders of magnitude.7,8 However, all evidence that the molecule is located in and emitted from these hot spots is indirect, based on expectations from theory and measured polarization responses.9
11 Although several groups have correlated electron microscopy images of SMSERS-active nanoparticle aggregates with spectral data from single molecules, none have been able to rigorously study the location of the molecule in the junction or experimentally map the enhanced signal associated with a specific hot spot due to the diffraction limit of light.9,10,12
15 Previous work has used super-resolution optical imaging to study the behavior of individual molecules in SERS hot spots, revealing that hot spots are significantly larger than expected.16,17 However, these previous studies lacked information about the associated structure of the aggregated nanoparticles that produced the SM-SERS signal. In this Letter, we use superresolution optical imaging to map the spatial origin of the SERS signal, and we correlate these SERS intensity maps with scanning electron microscopy (SEM) images to locate the origin of the Raman scattering within specific regions of the nanoparticle aggregates. SERS samples are prepared and analyzed on the optical and electron microscopes, as described in the Materials and Methods section. For each region of interest in a sample, we collect a series r 2011 American Chemical Society

of images (1000 total at 0.2 s integration) of the diffractionlimited spot associated with each SERS-active nanoparticle aggregate (see Supporting Information for sample images). For each frame in the image stack, we fit the point spread function (PSF) of the diffraction-limited spot to a two-dimensional Gaussian using the following equation Iðx, yÞ ¼ z0 þ I0 exp½
ð1=2Þ½ððx
x0 Þ=sx Þ

2

þ ððy
y0 Þsy Þ  2

ð1Þ

Here, I is the intensity for a given position (x,y) in space, z0 is the intensity of the background, I0 is the intensity of the Gaussian at the centroid position, sx,y is the standard deviation in x and y, respectively, and x0 and y0 are the centroid of the fit. The centroid allows us to track how the spatial origin of the SERS emission changes over time. In the case of multiple emitters within a single diffractionlimited spot, the centroid will be a superposition of multiple PSFs.18,19 Previously, we have handled this by exploiting the characteristic intensity fluctuations associated with SM-SERS and using a subtractive algorithm to spatially isolate each emitter.16 However, in this experiment, we observe two distinct spectral signatures, the R6G SERS, which turns on and off as expected, and a continuous spectral background from a secondary source, believed to be citrate (see Supporting Information for spectra).20
23 We observe fluctuations in the position of the Received: June 10, 2011 Accepted: July 1, 2011 Published: July 01, 2011 1766

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Figure 2. (A) SERS spatial intensity map and (B) SEM image of a silver colloid trimer. (C) Overlay of the spatial intensity map and SEM image. Figure 1. (A) SERS spatial intensity map and (B) SEM image of a silver colloid dimer. (C) Overlay of the spatial intensity map and SEM image.

background that prevent us from using the subtractive algorithm to isolate the origin of the R6G alone; however, we can fit the entire diffraction-limited spot to a single Gaussian and monitor changes in the centroid position with time as various emitters turn on and off.19 Because the SERS from the background is typically weak, the SERS centroid should shift toward the location of the hot spot when the R6G SERS turns on. We can confirm that these centroid shifts are due to R6G SERS with the simultaneously collected spectral data. After calculating the SERS centroid positions for all 1000 frames of an experiment, we calculate a 2-D histogram for the centroid data with a 0.1 pixel (4.6 nm) bin size and then calculate the mean intensity for all points within each bin to obtain a spatial intensity map, as shown in Figure 1A (see Supporting Information for the binning process). These spatial intensity maps show the spatial position of the most intense scattering events from a SERS-active nanoparticle aggregate, which we expect to be associated with a hot spot. Note that the size of the active area of the SERS centroid in Figure 1A is less than 50 nm, well below the diffraction limit of light yet larger than our theoretical resolution of 5 nm. The spatial intensity map shown in Figure 1A has a region of higher intensity on the right side of the image, and the scattering intensity decreases in gradient fashion as the centroid position shifts to the left, as also seen in previous work.16 After transferring the sample to the SEM and locating the same region of interest, we found that this aggregate was a dimer of silver nanoparticles (Figure 1B) with a diagonally oriented junction region. Theoretical studies of enhancement factors for aggregates of various shapes, sizes, interparticle spacing, and polarization angles are

prevalent in the literature and can be used to predict where regions of more intense scatter should occur.8,12,24
27 Given a single nanoparticle junction in a nanoparticle dimer, theory predicts that the region of most intense SERS will occur in that junction and that the SERS intensity should decay as the molecule moves away from the gap.8,28,29 By resizing the spatial intensity map to match the scale bar of the SEM image, the two pieces of data can be qualitatively overlaid, as shown in Figure 1C. The edge of the high-intensity region of the spatial intensity map is in excellent agreement with the orientation of the nanoparticle junction, and the gradient nature of the intensity, decreasing as the centroid moves further from the junction, matches expectations from theory.28,29 Although the region of highest intensity (single dark red bin in Figure 1C) does not align with the junction, this could be due to features present on the bottom surface of the colloids, such as an additional hidden nanoparticle, that scatter intensely but cannot be seen in the SEM images. This is due to the geometry of our system, in which optical and spectral data are collected in epi-illumination from the bottom side of the sample, whereas SEM images are collected from the top surface of the colloids. This issue could explain slight discrepancies in what theory would predict for hot spot geometry and what is seen in the spatial intensity map. Regardless of this single bin discrepancy, the spatial intensity map is an excellent fit for the colloid junction topography and orientation. Although we have chosen to place the spatial intensity map at the upper left of the junction due to the strong agreement between the shape of the nanoparticle and the shape of the spatial intensity map, it is important to note that this is a qualitative fit and it is possible that the SERS could originate from a lower portion of the junction. However, we do point out that the emission only originates from the left-hand side of the junction, suggesting that the molecule is confined on a single nanoparticle, consistent with our previous report.16 1767

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Figure 4. (A) SERS spatial intensity map and (B) SEM image of a silver colloid trimer. (C) Overlay of the spatial intensity map and SEM image.

Figure 3. Sequential SERS spatial intensity maps of the asymmetric trimer and overlaid SEM images (all scale bars are 50 nm).

The dimer in Figure 1 is an example of the type of structure that has been thoroughly modeled with theory, and consequently, the choice of overlay position was rather straightforward.8,25 The spatial intensity map shown in Figure 2A is more complex because there are multiple regions of increased intensity. SEM imaging reveals that this nanoparticle aggregate is a symmetric trimer of quasi-spherical silver colloids (Figure 2B). On the basis of theoretical predictions and our expectation that junction regions are associated with SERS hot spots, our observation of multiple hot spots in the spatial intensity map is consistent with this higher-order structure.30,31 As before, the spatial intensity map can be resized and overlaid with the SEM image, as shown in Figure 2C. In this overlay, we observe a region of strong SERS intensity (>750 intensity units) in the junction indicated with arrow 3. There are also excursions toward higher intensity in the two other junctions, indicated with arrows 1 and 2. However, we also observe mid-level intensity features (around 500
600 intensity units), indicated with arrows 4 and 5. We believe that these are due to junctions associated with a fourth hidden nanoparticle, the edge of which can be seen jutting from below arrow 3. In this case, the geometry of our imaging system prevents us from directly observing these junctions, as described above. The key feature to note for this overlay (Figure 2C) is that the high-intensity regions of the spatial intensity map exhibit spacing and gradient behavior consistent with theory and the structure of the nanoparticle.31 Although the effect of the fourth underlying nanoparticle on the spatial intensity map is unknown, the overlay is logical based on theory and our intuition of hot spot shape and size for a symmetric trimer. A third region of interest is shown in Figure 3 and reveals an asymmetric trimer. For this aggregate, three separate movies were collected, 200 s each, with a delay time of approximately 1

min between each movie. The spatial intensity maps and overlays are shown in the order of acquisition, with Figure 3A, B, and C corresponding to data from the first, second, and third movies, respectively. Initially, the upper junction region of the aggregate shows intense SERS scattering (Figure 3A), but as the experiment proceeds, SERS emission associated with the lower junction appears (Figure 3B and C). Importantly, the distance between the two regions of high SERS intensity matches well with the spacing between the two nanoparticle junctions. Although the spatial intensity maps make it appear as if the molecule begins in one junction and ends up in the other, we observe repeated exchange between the two locations (see Supporting Information, Figure S-8). Studies by Futamata et al. have attributed fluctuations in spectral intensity to diffusion of a single molecule in and out of a hot spot.32,33 One possibility for the observed exchange between the two hot spot junctions could be rapid diffusion of a single molecule on the surface of the aggregate. A second possibility is that we have two or more R6G molecules on the nanoparticle surface, and each is turning on and reporting on its associated hot spot at a given time (demonstrated in unpublished results). However, if at any point two molecules were emitting simultaneously, we would observe the centroid location as a superposition of the two junctions, and a region of strong SERS intensity would appear localized on the middle nanoparticle of the cluster. Although we do not observe this behavior, we cannot rule out the possibility of two separate molecules, each emitting at different times. A fourth region of interest, shown in Figure 4, has a spatial intensity map similar to that of Figure 1A, with only one region of higher intensity and similar intensity gradient behavior, suggesting the presence of a single junction region. However, when this aggregate was imaged in the SEM, it was found to contain another asymmetric trimer with two junction regions (Figure 4B). Although the overlay position might initially seem more complicated given the choice of junctions, the linearity, orientation, and size of the spatial intensity map agrees very well with the 1768

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The Journal of Physical Chemistry Letters geometry of the horizontal junction, as shown in Figure 4C. Placing the spatial intensity map in the vertically oriented junction region would not agree well with expectations from theory or with the geometry of the hot spot. We speculate that the lack of activity associated with the vertically oriented junction has two possible origins, (1) the junction is not SERS-active or (2) the molecule is not exploring that region of the nanoparticle. From the overlaid image, we see that the SERS centroid is localized exclusively on the leftmost nanoparticle, with no corresponding intensity on the center nanoparticle that shares the horizontally oriented junction. This lack of intensity suggests a possible barrier to diffusion across the nanoparticle junction, preventing the molecule from accessing the center or rightmost nanoparticle in the aggregate. A similar trend was also observed for the dimer in Figure 1, in which only the leftmost particle was explored, and the trimer in Figure 3, in which only the central nanoparticle has associated SERS intensity (associated with two separate junctions on either side of the nanoparticle). These examples could demonstrate a gap-dependent SERS phenomenon as predicted by numerous groups in which a molecule may only explore junction regions with a given spacing, resulting in spatial intensity maps that either fit into the junction region (as in Figure 2) or are positioned to one side of the junction (Figures 1, 3, and 4).30,34
36 In conclusion, we have shown the first correlated superresolution optical and SEM images of silver colloid SERS hot spots. The shape and gradient nature of the calculated spatial intensity maps show excellent agreement with theoretical predictions of field enhancement in nanoparticle junction regions. By overlaying the spatial intensity maps with the corresponding SEM images, we gain greater insight into the size and actual gradient intensity behavior of hot spots than that allowed with theory calculations alone. These correlated studies reveal that multiple junction regions in a SERS-active aggregate can support enhanced electromagnetic fields but also suggest that molecules may be confined to a single nanoparticle within an aggregate; both of these results have important implications for the design and fabrication of robust SM-SERS substrates.

’ MATERIALS AND METHODS SERS samples are prepared by incubating rhodamine 6G (R6G) in a solution of silver nanoparticles and then drop-casting the solution onto an indium tin oxide (ITO)-coated glass coverslip (see Supporting Information for full experimental details). The ITO coverslip is patterned with an alpha
numeric grid for registration between the optical and SEM images. Optical and spectral data are collected simultaneously using an inverted Olympus IX71 microscope with 532 nm excitation in epiillumination. The resulting SERS signal is split with a 50/50 beamsplitter, sending half of the signal to a PI ACTON SpectraPro 2500i spectrograph coupled to a Spec-10 camera for spectral acquisition, while the other half of the signal is passed to a Princeton Instruments ProEM 512 electron-multiplying CCD camera for SERS imaging. The structural information for nanoparticle aggregates is collected using an ultrahigh resolution Hitachi S-5500 SEM (30 kV accelerating voltage). ’ ASSOCIATED CONTENT

bS

Supporting Information. A more detailed explanation of the fitting process as well as associated spectra and intensity time

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traces for select regions of interest. This material is available free of charge via the Internet at http://pubs.acs.org.

’ AUTHOR INFORMATION Corresponding Author

*E-mail: [email protected]. Phone: 512.471.6488. Fax: 512.471.0985.

’ ACKNOWLEDGMENT We thank Spherotech for the generous gift of fluorescent nanospheres used as alignment markers. We also thank the National Science Foundation (Grant No. 0821312) for funding the Hitachi S-5500 scanning electron microscope/scanning transmission electron microscopy used in this work and Texas Materials Institute for supporting this facility. This material is 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. ’ REFERENCES (1) Nie, S.; Emory, S. R. Probing Single Molecules and Single Nanoparticles by Surface-Enhanced Raman Scattering. Science 1997, 275, 1102–1106. (2) Kneipp, K.; Wang, Y.; Kneipp, H.; Perelman, L. T.; Itzkan, I.; Dasari, R. R.; Feld, M. S. Single Molecule Detection Using SurfaceEnhanced Raman Scattering (SERS). Phys. Rev. Lett. 1997, 78, 1667–1670. (3) Etchegoin, P. G.; Le Ru, E. C. A Perspective on Single Molecule SERS: Current Status and Future Challenges. Phys. Chem. Chem. Phys. 2008, 10, 6079–6089. (4) Qian, X.-M.; Nie, S. M. Single-Molecule and Single-Nanoparticle SERS: From Fundamental Mechanisms to Biomedical Applications. Chem Soc. Rev. 2008, 37, 912–920. (5) Moskovits, M.; Tay, L.-L.; Yang, J.; Haslett, T. SERS and the Single Molecule. Top. Appl. Phys. 2002, 82, 215–227. (6) Dieringer, J. A.; Lettan, R. B., II; Scheidt, K. A.; Van Duyne, R. P. A Frequency Domain Existence Proof of Single-Molecule SurfaceEnhanced Raman Spectroscopy. J. Am. Chem. Soc. 2007, 129, 16249– 16256. (7) Michaels, A. M.; Nirmal, M.; Brus, L. E. Enhanced Raman Spectroscopy of Individual Rhodamine 6G Molecules on Large Ag Nanocrystals. J. Am. Chem. Soc. 1999, 121, 9932–9939. (8) Hao, E.; Schatz, G. C. Electromagnetic Fields Around Silver Nanoparticles and Dimers. J. Chem. Phys. 2004, 120, 357–366. (9) Shegai, T.; Li, Z.; Dadosh, T.; Zhang, Z.; Xu, H.; Haran, G. Managing Light Polarization Via Plasmon
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