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May 14, 2010 - Darkfield imaging of single AuNRs settled onto the glass slide surface were performed using an upright microscope (Nikon 80i) equipped ...
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Anal. Chem. 2010, 82, 5268–5274

Three Dimensional Orientational Imaging of Nanoparticles with Darkfield Microscopy Lehui Xiao, YanXia Qiao, Yan He,* and Edward S. Yeung* Biomedical Engineering Center, College of Chemistry and Chemical Engineering, State Key Laboratory of Chemo/ Biosensing and Chemometrics, Hunan University, Changsha, 410082, P.R. China The complete three-dimensional orientations of single gold nanorods (AuNR) were successfully resolved by using a standard optical darkfield microscope through deciphering the field distribution pattern in the slightly defocused darkfield images. The resulting images depend on the aspect ratio of the AuNR, the numerical aperture of the objective, the defocusing distance, and the polarization direction of the incident radiation. Interpretation of the observed images is facilitated by comparing them with a series of simulated images with different parameters. The experimental data matched well with the simulated results, and the reliability of this technique was further verified with polarization modulation experiments. Since deconvolution can be performed off-line after the images are recorded, this approach essentially allows video-rate data acquisition. The convenient, reliable and rapid angleresolving capability should enable broad applications in imaging studies in many scientific fields. Recent achievements in fabrication, manipulation and characterization of metal particles at the nanometer scale have generated renewed interests from physicists, chemists and biologists in localized surface plasmon resonances (LSPR).1 LSPR are collective coherent oscillations of the conduction band electrons on the surface of plasmon resonant nanoparticles (PRPs). The resonance peak position, intensity and the peak width of PRPs are extremely sensitive to the factors which could induce any slight variations in the charge distribution on the nanoparticle surface, i.e. the material composition, the shape and size of the metal nanoparticles, the dielectric constant of the surrounding environment, and so on.1,2 Another distinguishing feature of PRPs is that their absorption and scattering cross sections are magnitudes larger than dye molecules. For example, for gold nanospheres with diameter around 40 nm, the cross sections are determined to be 5 to 6 orders of magnitude larger than those of conventional transitions.3 This leads to pronounced field enhancements in Rayleigh scattering, Raman scattering and fluorescence from PRPs and makes them some of the most popular optical imaging contrast agents in recent years.4-6 Compared with dye molecules, * Authors to whom e-mail may be sent. E-mail: [email protected]; [email protected]. (1) Willets, K. A.; Duyne, R. P. V. Annu. Rev. Phys. Chem. 2007, 58, 267–297. (2) Link, S.; El-Sayed, M. A. J. Phys. Chem. B 1999, 103, 8410–8426. (3) Jain, P. K.; Lee, K. S.; El-Sayed, I. H.; El-Sayed, M. A. J. Phys. Chem. B 2006, 110, 7238–7248. (4) Nie, S.; Emory, S. R. Science 1997, 275, 1102–1106.

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the significant advantages of PRPs are that they do not photobleach or blink, and do not optically saturate at reasonable exciting intensities.5 Moreover, the surface property of the PRPs could be easily modulated via surface chemistry. Therefore, PRPs are considered to be the most promising alternatives for optical imaging and biosensing.1,7 Meanwhile, a great number of high-throughput optical microscopic imaging methodologies have also been developed to detect and track single PRPs, such as darkfield imaging at the plasmon resonance frequency, differential interference contrast microscopy, total internal reflection microscopy, and photothermal interference contrast imaging.5,8-12 These methods are generally based on the detection of Rayleigh scattering or absorbed light from PRPs and usually only positional information could be extracted from the recorded CCD image. On the other hand, recent studies have demonstrated that the magnitude of field enhancement (surface enhanced Raman or fluorescence) is strongly dependent on the spatial orientation of anisotropic PRPs.13,14 Moreover, vast biological applications have also demonstrated that the spatial orientation of PRPs is of great importance for scientists to understand certain vital biological mechanisms, such as the conformation fluctuation dynamics of proteins, the rotational motion of ATPase, the heterogeneous local structure deformations of the cell membrane, or the interaction force between proteins and cell membranes, etc.15-19 Therefore, extracting the orientational information from (5) Schultz, S.; Smith, D. R.; Mock, J. J.; Schultz, D. A. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 996–1001. (6) Aslan, K.; Gryczynski, I.; Malicka, J.; Matveeva, E.; Lakowicz, J. R.; Geddes, C. D. Curr. Opin. Biotechnol. 2005, 16, 55–62. (7) Schultz, D. A. Curr. Opin. Biotechnol. 2003, 14, 13–22. (8) So ¨nnichsen, C.; Franzl, T.; Wilk, T.; Plessen, G. v.; Feldmann, J. Phys. Rev. Lett. 2002, 88, 077402. (9) So ¨nnichsen, C.; Geier, S.; Hecker, N. E.; Plessen, G. v.; Feldmann, J.; Ditlbacher, H.; Lamprecht, B.; Krenn, J. R.; Aussenegg, F. R.; Chan, V. Z.H.; Spatz, J. P.; Mo ¨ller, M. Appl. Phys. Lett. 2000, 77, 2949–2951. (10) Boyer, D.; Tamarat, P.; Maali, A.; Lounis, B.; Orrit, M. Science 2002, 297, 1160–1163. (11) Jacobsen, V.; Stoller, P.; Brunner, C.; Vogel, V.; Sandoghdar, V. Opt. Express 2006, 14, 405–414. (12) Sun, W.; Wang, G.; Fang, N.; Yeung, E. S. Anal. Chem. 2009, 81, 9203–9208. (13) Ming, T.; Zhao, L.; Yang, Z.; Chen, H.; Sun, L.; Wang, J.; Yan, C. Nano Lett. 2009, 9, 3896–3903. (14) Wei, H.; Hao, F.; Huang, Y.; Wang, W.; Nordlander, P.; Xu, H. Nano Lett. 2008, 8, 2497–2502. (15) Kukura, P.; Ewers, H.; Mu ¨ ller, C.; Renn, A.; Helenius, A.; Sandoghdar, V. Nat. Methods 2009, 6, 923–927. (16) Toprak, E.; Enderlein, J.; Syed, S.; McKinney, S. A.; Petschek, R. G.; Ha, T.; Goldman, Y. E.; Selvin, P. R. Proc. Natl. Acad. Sci. U.S.A. 2005, 103, 6495–6499. (17) Spetzler, D.; York, J.; Daniel, D.; Fromme, R.; Lowry, D.; Frasch, W. Biochemistry 2005, 45, 3117–3124. 10.1021/ac1006848  2010 American Chemical Society Published on Web 05/14/2010

anisotropic PRPs seems to be a universal problem that needs to be solved urgently. So far, only a few methods have been reported to have the capability to determine the orientation of anisotropic PRPs in situ and most of them are derived from orientation imaging techniques for fluorescent molecules.20-27 One of them is polarization imaging which uses dark-field microscopy when combined with a birefringent crystal to split the perpendicular and parallel polarized scattering light from anisotropic PRPs.24,28 In that setup, only relative 2D orientation information were effectively obtained while the out-of-plane angle was completely obscured, which could be a vital parameter in some complex processes. Another approach is to attach a single gold nanoparticle to the end of a sharp glass fiber tip and then illuminate it with white light.29,30 By precisely collecting the scattering light and plasmon spectra as a function of the polarization angle of the incident light, the ellipticity and the angular information of the gold nanoparticle could be resolved. However, the time resolution of this method is very limited such that one cannot simultaneously resolve a large number of randomly orientated PRPs. Another candidate is imaging the PRPs with confocal microscopy through higher order mode laser excitation.22,31 This method is initially developed to determine the dipole orientation of fluorescent dye molecules and needs sophisticated excitation sources and complex laser mode modulation.22,32 The protocol is also time-consuming which limits its application in bioscience or other dynamical investigations. Therefore, it is important to develop a convenient, inexpensive, in situ and high throughput method to extract the 3D orientational information from large numbers of PRPs simultaneously. In this article, we present a novel technique and proof of concept experiments to demonstrate that gold nanorod (AuNR) 3D orientations can be obtained with standard optical darkfield microscopes. The spatial angles of AuNRs were conveniently derived through deciphering the field distribution pattern in the defocused darkfield image. The methodology reported here is highly reliable and offers great improvements in comparison to previously reported methods, such as fast full 3D angle resolving capability, freedom from photo(18) Stone, J. W.; Sisco, P. N.; Goldsmith, E. C.; Baxter, S. C.; Murphy, C. J. Nano Lett. 2007, 7, 116–119. (19) Pierrat, S.; Hartinger, E.; Faiss, S.; Janshoff, A.; So¨nnichsen, C. J. Phys. Chem. C 2009, 113, 11179–11183. (20) Jasny, J.; Sepiol, J. Chem. Phys. Lett. 1997, 273, 439–443. (21) Sepiol, J.; Jasny, J.; Keller, J.; Wild, U. P. Chem. Phys. Lett. 1997, 273, 444–448. (22) Sick, B.; Hecht, B.; Novotny, L. Phys. Rev. Lett. 2000, 85, 4482–4485. (23) Dickson, R. M.; Norris, D. J.; Moerner, W. E. Phys. Rev. Lett. 1998, 81, 5322–5325. (24) Harms, G. S.; Sonnleitner, M.; Schutz, G. J.; Gruber, H. J.; Schmidt, T. Biophys. J. 1999, 77, 2864–2870. (25) Bo ¨hmer, M.; Enderlein, J. J. Opt. Soc. Am. B 2003, 20, 554–559. (26) Dedecker, P.; Muls, B.; Deres, A.; Uji-i, H.; Hotta, J.-i.; Sliwa, M.; Soumillion, J.-P.; Mullen, K.; Enderlein, J.; Hofkens, J. Adv. Mater. 2009, 21, 1079– 1090. (27) Patra, D.; Gregor, I.; Enderlein, J.; Sauer, M. Appl. Phys. Lett. 2005, 87, 101103. (28) So ¨nnichsen, C.; Alivisatos, A. P. Nano Lett. 2005, 5, 301–304. (29) Kalkbrenner, T.; Hakanson, U.; Schadle, A.; Burger, S.; Henkel, C.; Sandoghdar, V. Phys. Rev. Lett. 2005, 95, 200801–200804. (30) Kalkbrenner, T.; Hakanson, U.; Sandoghdar, V. Nano Lett. 2004, 4, 2309– 2314. (31) Failla, A. V.; Qian, H.; Qian, H.; Hartschuh, A.; Meixner, A. J. Nano Lett. 2006, 6, 1374–1378. (32) Lieb, M. A.; Zavislan, J. M.; Novotny, L. J. Opt. Soc. Am. B 2004, 21, 1210– 1215.

bleaching, highly parallel data acquisition, convenient and inexpensive experimental setup, and so on. EXPERIMENTAL SECTION Reagents and Materials. AuNRs with axial diameter 25 nm and longitudinal length 60 nm were purchased from Nanopartz (Loveland, CO). In a typical experiment, 5 µL of diluted AuNR solution was dropped onto a precleaned glass slide and covered with a 22 mm × 22 mm coverglass immediately. To avoid water evaporation, edges of the cover glass were sealed with enamel carefully. Apparatus. Darkfield imaging of single AuNRs settled onto the glass slide surface were performed using an upright microscope (Nikon 80i) equipped with a two-port digital imaging head. The illumination light was provided by a 100 W halogen lamp and focused onto the sample with an oil immersion darkfield condenser (NA ) 1.20-1.43). An NA adjustable (from 0.7 to 1.25) 60× darkfield objective was used to collect the scattered light from AuNRs. The darkfield image was acquired with a monochrome CCD camera (CoolSnap HQ2, Photometrics, U.S.A.) that was mounted on the front port of the microscope. To confirm that light spots on the monochrome CCD image were real scattering AuNRs, a color CCD camera (DP72, Olympus) was mounted on the back port of the Nikon 80i microscope to acquire the color image simultaneously. A motorized rotational stage (SGSP-40YAM, Sigma Koki) was coupled to the fine adjustment knob of the microscope to precisely control the stage position at the z-direction with a resolution of 0.69 nm. Image Processing. All of the images were processed with ImageJ (http://rsbweb.nih.gov/ij/) or Matlab. The computer simulations were performed using Matlab. RESULTS AND DISCUSSION Scattering Electrical Field Strength of Oscillation Dipoles from AuNRs. With our darkfield imaging microscope, PRPs were evenly illuminated with randomly polarized light that was tightly focused by a high NA darkfield oil condenser. The measured bright scattering spot on the darkfield image is the time-averaged Poynting vector along the optical axis (z direction)33,34

〈S〉 )

| |

1/2 cεm ∧ 1∧ ez Re{E(scat) × H*(scat)} ) e E 2 8π z (scat)

2

(1)

where εm is the dielectric constant of the medium, c is the speed of light in vacuum, and E(scat) denotes the scattering electric field from the AuNR. According to the electrostatic approximation, plasmon oscillations from anisotropic PRPs can be simplified as multi-independent-oscillation dipoles.34,35 In the case of AuNR (ellipsoid nanoparticle), the electron oscillation has been confirmed to take place at three orthogonal directions that are dependent on the polarization angle of the excitation light.35 Oscillation along the long principal axis is defined as a longitudinal mode, and the other perpendicular oscillations are called trans(33) Hellen, E. H.; Axelrod, D. J. Opt. Soc. Am. B 1987, 4, 337–350. (34) Bohren, C. F.; Huffman, D. R. Absorption and Scattering of Light by Small Particles; Wiley Intersciene: New York, 1983. (35) Link, S.; Mohamed, M. B.; El-Sayed, M. A. J. Phys. Chem. B 1999, 103, 3073–3077.

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Figure 1. (Left) Calculated oscillation strength of the longitudinal mode and the transverse modes as a function of the AuNR aspect ratio. The wavelength of illumination is set to be 600 nm. (Right) Calculated scattering spectra of AuNRs with different aspect ratios according to the Gans theory.35

verse modes that vibrate along the short axes. Strength of the induced oscillation dipole moment is determined by the projection of the external electromagnetic field and the polarizability of PRPs along the corresponding dipole axis and can be quantitatively expressed as34 Pj ) εmRjEj

(2)

where Ej is the projection of the excitation electric field along the dipole axis and is equal to E0 exp(ikj - iωt)êj. Rj defines the polarizability of the nanorod along each principal axis Rj ) 4πxyz

εr - εm 3εm + 3Lj(εr - εm)

(3)

where Lj is a geometrical factor that only depends on the aspect ratio of the nanorod and εr is the real part of the bulk gold dielectric function. In the case of x > y ) z, Lj is determined by Lx )

1+R 1 - R2 1 ln -1 2 2R 1 -R R

)

(

(4)

where R)

1 - ( xy )

2

The other two geometrical factors can be deduced from Ly ) Lz ) (1 - Lx)/2. These equations indicate that for a homogeneous static excitation, the relative strengths of oscillation dipoles within single PRPs are purely determined by the geometric factor Lj. Since the orthogonal oscillation dipoles are considered to be absolutely independent, the overall scattering electrical field emanating from the nanorod could be quantified through linear superposition of the three scattering fields, one from each oscillation dipole, E(scat) )

∑E

(scat)j)E(scat)x

+ E(scat)y + E(scat)z

x,y,z

where 5270

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E(scat)j )

∧ eik(r-z) ik3 ∧ e × (er × Pj) -ikr 4πεm r

(5)

From eqs 1 to 5, we could note that the observed darkfield image contains the complete unresolved spatial orientation information from the three independent orthogonal oscillation dipoles. As a result, the most direct optical imaging method to map the orientation of AuNR is to characterize the plasmon intensity distribution from each scattering dipole. Figure 1 illustrates the calculated scattering intensity of the three orthogonal oscillation dipoles from an AuNR as a function of aspect ratio when illuminated by parallel polarized light with wavelength at 600 nm (the peak position of the microscope light source). It is clear that the magnitude of the longitudinal oscillation increases rapidly with increasing aspect ratio while the transverse modes nearly keep constant. The scattering intensity of the longitudinal mode rapidly reaches a maximum when the aspect ratio of the nanorod is about 2.2 and then decreases quickly but is still much larger than the strength of the transverse modes (see the intensity ratio curve). This is because the maximum longitudinal plasmon resonance of AuNR with an aspect ratio of 2.2 is located near 600 nm. Any slight deviation from this resonance peak would strongly disturb the longitudinal scattering strength. In other words, the strength contribution from each dipole greatly varies when the morphology of the PRPs is changed. Therefore, the field distribution on the darkfield image reflects not only the three-dimensional information but also the topographic message from the PRPs. Effect of Objective NA Value on the Defocused Darkfield Image. In the conventional imaging mode that probes the specimen in the sharp focal plane, even though the full angular information is completely contained within the time averaged scattering Poynting vector, the three-dimensional orientation of the AuNR cannot be resolved because the scattered light from all the dipoles on the focal plane are focused into a single Airy disk. However, if an aberration (slight shift of the dipole away from the focal plane) is deliberately introduced to the imaging system, for an AuNR with a specific orientation the angle of each oscillation dipole can be resolved according to the intensity

Figure 2. Images in the upper panel are the measured darkfield images of AuNR with defocusing distance varying from 0.3 to 1.3 µm. The magnification of the objective is set to 60 with NA ) 1.2. Each pixel on these images represents 0.1 µm. The corresponding simulated darkfield images are illustrated in the lower panel. Orientation of AuNR for the simulation experiment is adjusted until it accords well with the experiment result.

distribution of the squared scattering electric field.33,36 As a result, for a certain defocusing distance, one could readily decipher the angle of an AuNR by first mapping a precise relationship between its orientation and the squared scattering electric field distribution pattern in the image space. Here, we successfully implemented this concept with an ordinary optical darkfield microscope, and the comprehensive experimental verifications are presented as follows. The detailed electromagnetic theory of scattering electric field distribution on the CCD image as a function of the in-plane angle, out-of-plane angle and defocusing depth from the AuNR can be unambiguously deduced from refs 25 and 27. According to the discussion above, the orientational information of an AuNR is uniquely reflected in the term of its field distribution in the image space. Therefore, the major problem is to make sure that each component could be well-recorded and -resolved on the CCD image. Since the field strength decreases rapidly when the imaging plane is shifted away from the focal plane, the optimum defocusing distance to decipher the AuNR angle that does not degrade the image quality is where the field pattern can be resolved initially. Figure 2 shows the measured and simulated darkfield image of a randomly selected AuNR with various defocusing distance while keeping the NA value at 1.2. It is clear that, under this imaging condition, the optimum defocusing distance is 0.9 µm. At a fixed defocusing distance and magnification, the overall light collection efficiency and the diffraction pattern of the focused light are principally determined by the NA value of the objective,36,37 where NA ) n sin θ, and n is the refractive index of the object space. Figure S1 (Supporting Information) shows the simulated scattering images from an AuNR (aspect ratio equals about 2.5) with different NA values while the magnification and the defocusing distance are fixed. When the NA is gradually changed from 0.7 to 1.0 (the maximum collection angle of the objective ranges from 31.7° to 48.7°, which is much smaller than the critical angle of 61.6° at the interface), the (36) Gibson, S. F.; Lanni, F. J. Opt. Soc. Am. A 1991, 8, 1601–1613. (37) Enderlein, J. Opt. Lett. 2000, 25, 634–636.

Figure 3. Left column depicts the measured darkfield images of an AuNR captured by a 60× objective with different NA values. Right column depicts the corresponding simulated darkfield patterns.

darkfield images show typical well-defined Airy disk shape even though they are captured in the defocused mode. Hence, it is impossible to deconvolute the AuNR 3D angle information from them. As the NA value becomes >1, the Airy disk starts to distort gradually, and a clear double-lobe structure appears. The intensity distributions on these images are no longer symmetric as are the Gaussian profiles in the images for NA < 1. Consequently, useful angular information could be readily extracted according to the characteristic field distribution pattern. When NA reaches 1.2, the maximum collection angle of the objective becomes slightly larger than the critical angle. Further increase in the NA value would not improve the angle-resolving capability but would adversely increase the background signal. These simulation results were verified to match exactly the experimental results (Figure 3). Effect of AuNR Aspect Ratio on the Defocused Darkfield Image. As demonstrated above, the relative contribution from each oscillation dipole is purely determined by the aspect ratio. As a result, the defocused darkfield image should also reflect the morphology information of the AuNR. Figure 4 illustrates a sequence of simulated defocused darkfield images of AuNR with different aspect ratios. In the case of the nanorod degenerating into a spherical structure, the field distribution of the darkfield image is symmetrically spread and displays a doughnut shape. This is because the contribution from each dipole is equal. As the aspect ratio increases, the contrast of the lobes becomes much clearer and the edge becomes much sharper. This is in good agreement with the calculated intensity ratio spectra where the strength of longitudinal oscillation significantly dominates the transverse oscillations as the structure of PRPs is gradually elongated. This indicates that, in the ideal case, the aspect ratio of AuNR could also be determined according to the sharpness of the field patterns as shown in Figure 4. Analytical Chemistry, Vol. 82, No. 12, June 15, 2010

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Figure 4. (Left) Simulated darkfield images of gold nanoparticles with aspect ratios of 1.0, 1.5, 2.0 and 2.5, respectively. The magnification of the objective is set to 60 with NA 1.2. The defocusing distance is 0.9 µm for all of the images. (Middle) Illustration of spatial orientation of each particle. (Right) 3D intensity distribution of the field patterns from the corresponding darkfield images in the left panel.

Angle-Resolved Darkfield Imaging of AuNRs. A map of simulated darkfield images from an AuNR (aspect ratio 2.5) with various 3D orientations is shown in Figure S2 (Supporting Information). From these images, we can conclude that each specific angle is correlated with a unique, defocused darkfield image. They vary noticeably once the angle of the nanorod is rotated. As a result, the random orientations of AuNRs on a substrate could be easily determined by referring to their corresponding fingerprint map. In order to further verify the reliability of this technique, we compared the result with that from polarization modulation.13 It is well-known that the 2D in-plane orientation (the projection of the oscillation dipole onto the image plane) of AuNRs on a substrate could be accurately determined via modulating the polarization angle of the illumination light. The quantitative relationship between the scattering intensity from the AuNR, Iscat, and the cross angle, θ (the angle between the longitudinal axis of AuNR and the transmission axis of the polarizer), is determined to be Iscat ≈ Io cos2 θ, where Io is the maximum value of the scattering light from the AuNR.38 Figure 5 shows the measured and the corresponding simulated darkfield image of a randomly selected AuNR on a glass slide surface. The typical red scattered light on the color CCD image further confirmed that the scattering is from a well-defined single AuNR (38) Born, M.; Wolf, E. Principles of Optics: Electromagnetic Theory of Propagation, Interference and Diffraction of Light, 7th ed.; Cambridge University Press: Cambridge, England, 1999.

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Figure 5. (Upper left) Darkfield image of AuNR measured by a 60× objective with NA 1.2. The defocusing distance is carefully adjusted to 0.9 µm using a motorized rotational stage. (Upper right) Corresponding best-fit simulated darkfield image. (Lower left) Blue curve in the polar plot is the calculated result according to the fitted inplane angle. Red circles are the measured results through polarization modulation. (Lower right) Determined 3D orientation of the AuNR is illustrated as a red rod. The blue line is the corresponding in-plane projection.

(Figure S3). Through fitting the field distribution pattern, the 3D angle of the AuNR is measured to have a polar angle of 66° and an azimuthal angle of 41°. This measured in plane projection angle perfectly matches that obtained through polarization modulation as demonstrated in the polar plot. Although accurate in-plane projection information can be deduced from polarization modula-

Figure 6. Defocused darkfield image of AuNRs positioned on a glass slide surface.

Figure 7. (Left column) Measured darkfield images from four randomly selected AuNRs on a glass slide. The imaging condition is the same as that in Figure 5. The other columns from left to right are the best-fit simulated darkfield images, the 3D intensity distribution plots and 3D orientation models of the corresponding AuNRs, respectively. The contour plots and blue dots on the 3D intensity distribution plots are the measured values, depicting a good match with the simulated patterns.

tion, the tilt angle information is lost and cannot be resolved directly from the scattering curve. Therefore, the method introduced in this work is demonstrated to be practically more reliable and much more informative. At the same time, it also provides the high throughput capability of wide field optical microscopy. The approach can thus be utilized to simultaneously track and monitor multiple dynamic events with fast data acquisition rate due to the strong scattering signal from PRPs. Since surfaces play a key role in chemical reactions and in chromatographic separations, as an initial demonstration of an application of this method, the surface spatial heterogeneity of a glass slide was studied here. A typical defocused darkfield image of randomly orientated AuNRs on a glass slide surface is shown in Figure 6. The diverse field distribution patterns indicate that the orientations of AuNRs are highly varied. Four AuNRs are randomly selected, and their 3D angles are subsequently determined by fitting with the simulated fingerprint patterns (Figure 7). The measured diverse nonzero polar angles indicate that the glass slide surface flatness is seriously nonuniform. AFM measurements on these glass slides (data not shown) confirm height variations in the 50 nm range (length of the AuNRs) together with larger pits and scratches within a 40 × 40 µm area. In addition, CTAB and salts that may be present in the AuNR preparation can influence the attachment of AuNR to the glass surface. Such in

situ spatial heterogeneity may provide valuable information for understanding the anomalous diffusion behavior of single biomolecules at the liquid/solid interface.39,40 Future work in this direction is being pursued in our laboratory.

CONCLUSION In summary, we have introduced a novel widefield optical imaging method to effectively record the 3D orientation angles of AuNRs with high reliability. Through deconvolution of the field distribution pattern from each oscillation dipole on the darkfield image, the spatial information of AuNRs was readily obtained. Since deciphering the images can be done off-line at a later time, video-rate date acquisition of multiple AuNRs is possible. Compared with previous reports, the method described here provides considerable improvements such as fast full 3D angle resolution capability, parallel data acquisition, convenient and inexpensive experimental setup, etc. The first two features should be particularly beneficial for the study of molecular adsorption phenomenon and single-cell in situ dynamics. (39) Xu, X.-H.; Yeung, E. S. Science 1997, 275, 1106–1109. (40) Schuster, J.; Cichos, F.; Wrachtrup, J.; von Borczyskowski, C. Single Mol. 2000, 4, 299–305.

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ACKNOWLEDGMENT This work was supported by NSFC 20605008, NSFC 20975036, Program for New Century Excellent Talents in University and Hunan University 985 Fund. E.S.Y. thanks the Ames Laboratory for partial support of this work. SUPPORTING INFORMATION AVAILABLE Additional images of gold nanorods acquired using the 60× objective with different NA values and the corresponding simulated results, simulated fingerprint map of gold nanorods with

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various orientations, color CCD image of gold nanorod and the corresponding polarization modulation result, and a typical darkfield image of gold nanorods on a glass slide. This material is available free of charge via the Internet at http://pubs.acs.org.

Received for review March 16, 2010. Accepted May 3, 2010. AC1006848