NANO LETTERS
Gold Nanorods as Novel Nonbleaching Plasmon-Based Orientation Sensors for Polarized Single-Particle Microscopy
2005 Vol. 5, No. 2 301-304
Carsten So1 nnichsen and A. Paul Alivisatos* Department of Chemistry, UniVersity of California, Berkeley, California 94720, and Materials Sciences DiVision, Lawrence Berkeley National Laboratory, Berkeley, California 94720 Received November 18, 2004; Revised Manuscript Received December 7, 2004
ABSTRACT By monitoring the polarized light scattering from individual gold nanorods in a darkfield microscope, we are able to determine their orientation as a function of time. We demonstrate time resolution of milliseconds and observation times of hours by observing the two-dimensional rotational diffusion of gold rods attached to a glass surface. The observed orientational diffusion shows a fast component of about 60 ms and “sticky times” of seconds. The large signal-to-noise ratio, chemical and photochemical stability, fast time response, and small size of these gold nanorods make them an ideal probe for orientation sensing in material science and molecular biology.
In the growing field of single-molecule spectroscopy, there has been considerable recent interest in optical probes for nanoscale orientation sensing. The currently employed technique is based on measuring the polarization of anisotropic fluorescent probes1-10 or the lateral position of micrometer-sized beads.11,12 Local orientation sensors are important in material research, e.g., the study of liquids in confined spaces,13 liquid crystal orientation,14 and the structure of glasses and polymers.15 In biomolecular research, monitoring orientation changes and rotations of biomolecules during their functional task have explained the mechanism of molecular motors12,16 and helped to explain the mechanical properties of DNA.11 A major drawback of fluorescent labels is the limited observation time due to photobleaching (organic dyes) and the erratic signal due to blinking. Monitoring the lateral position of micrometer-sized beads with nanometer precision offers greater photostability and no blinking. A drawback of this technique is the size of such beads, which might influence the behavior of the attached biomolecules and limit the time resolution due to their viscous drag. Also, the observable movements are restricted to lateral changes. Ideally, one would like to combine the advantages of these two methods. Metal nanoparticles offer an alternative label to fluorescent dyes or quantum dots.17,18 Gold and silver nanoparticles show strong light scattering at the plasmon resonance wavelength due to the collective oscillation of their conduction electrons. This light scattering is orders of magnitude greater than that * Corresponding author. E-mail:
[email protected]. 10.1021/nl048089k CCC: $30.25 Published on Web 12/24/2004
© 2005 American Chemical Society
of a nonmetallic object of the same size. By imaging only scattered light from a sample in darkfield12,19,20 or total internal reflection microscopy,21 it is possible to image particles down to 20 nm in size. Alternatively, their absorption can be used in photothermal imaging.22 Especially gold nanorods have been shown to be extremely strong light scatteres due to the combination of lightning rod effect and the suppression of interband damping.20 In addition, it was shown that light scattered off gold rods is strongly polarized along the long axis,20,23 making gold nanorods in principle an ideal orientation probe. We test this concept by monitoring the time dependent polarized light scattering of rotating single gold nanorods. By carefully controlling ionic strength and pH, we are able to attach gold nanorods loosely to the surface of a glass flow cell and still allow two-dimensional rotational motion. We demonstrate time resolutions down to a few milliseconds and observation times of hours, several orders of magnitude better than any fluorescent label. We anticipate further improvement by employing laser illumination, lock-in techniques, and faster detectors. The method should therefore find applications in a wide range of fields where local orientation changes are of interest. In our experiments we use gold nanorods prepared by a seed growth method derived from Jana et al.24 Seeds are prepared by reducing 10 mL of an aqueous solution containing 0.25 mM gold tetrachloride (HAuCl4) in 0.1 M hexadecyltrimethylammoniumbromide (CTAB) by adding 0.6 mL of 0.01 M sodium borohydride (NaBH4). A 12 µL portion of these seeds is added to 10 mL of a growth solution consisting of 0.5 mM HAuCl4 in 0.1 M CTAB mixed with
Figure 1. (a) Darkfield setup used to image the gold nanorods. Illumination is through a darkfield oil immersion condenser; only scattered light is collected by an air objective, and a bifringent calcite crystal splits the light into orthogonal polarization directions. (b) A TEM image of the gold nanorods. (c) Darkfield image of gold nanorods (averaged over 30 s). Most rods appear as two dots separated a few pixels horizontally. The horizontal splitting is caused by the bifringent crystal, which displaces one polarization direction a few micrometers with respect to the orthogonal polarization direction. (The few single dots are gold rods stuck in a position parallel to one of the two polarization axes.) The software automatically picks out spots which show temporal fluctuations (red squares).
70 µL of 0.0788 M ascorbic acid. After 10-20 min a strong color change indicates the formation of gold rods. The gold rods are relatively homogeneous in size and shape (Figure 1b) and can be used as prepared without further purification steps. Their size, as deduced from TEM images, is approximately 25 nm × 60 nm with less than 20% dispersion. The principle of the darkfield setup is shown in Figure 1a. The sample is illuminated by white light from a 100 W Tungsten lamp through an oil immersion darkfield condenser.23 The scattered light is collected by a 40× objective (NA 0.65) and images recorded with a fast, intensified camera (Cascade, Roper Scientific). A bifringent calcite crystal introduced in the light path splits two orthogonal polarization directions for each gold rod into two spots on the detector to allow their simultaneous intensity monitoring (Figure 1c). The sample cell consists of a thin, flat glass capillary (0.1 mm × 2 mm × 100 mm) connected to PET tubing, which allows us to flush in various fluids. A 1 µL portion of rod solution is diluted in 100 µL ddH2O and flushed into the sample cell. After letting the gold rods settle for 5 min, the particles are washed away by flushing with 1 mL of ddH2O containing 50 mM NaCl. Some particles remained attached to the surface after flushing. The salt increased the number of gold rods attached to the surface considerably. After adjusting the pH of the solution to 8, about 50% of the rods start to rotate on the surface, which is observed as a beating in the intensity between the two orthogonal polarizations (see Supporting Information). The particles remain in aqueous environment at all times. The camera records a movie of the sample area with a frame rate of 30 Hz for the full frame (512 × 512 pixels) and up to 300 Hz for a smaller area. The 302
Figure 2. Upper graph: Time traces of the intensity observed for the two orthogonal polatization channels of an individual gold nanorod (green/red). The intensity adds up to an almost constant total intensity (black), as expected for two-dimensional rotations. From the relative intensity in the two channels, the orientation (angle) of the rod may be computed (lower graph). The orientation direction is ambiguous with respect to -45/45 degrees (insets).
resulting datasets are processed using code written in Matlab (The Mathworks Inc.). The software automatically identifies bright spots, collects the time traces of their intensity, and sorts out those which show temporal fluctuations in intensity (Figure 1c). An example of a time trace of one gold rod is shown in Figure 2a. The two polarization channels are shown in different colors and their sum in black. The total intensity remains constant within the noise level; the intensity fluctuates only between the two polarization directions. This Nano Lett., Vol. 5, No. 2, 2005
Figure 3. Left: Time traces of orientation (angle) for four representative particles (A,B,C,D). Particle A starts off confined in one orientation, then at later times rotates more rapidly. Particle B shows various long “sticky periods” of several seconds duration. Particle C shows only very few “sticky times”, whereas particle D seems to be freely rotating. Right: the corresponding autocorrelation curves show significant correlation for particle A and B on the second time scale from the “sticky periods”. The correlation for particles C and D fall off exponentially with a time constant of 60-120 ms. The oscillations at time >500 ms are artifacts introduced by the limited observation time.
observation is consistent with a two-dimensional confinement of the gold rods to the surface of the sample chamber. If the rods were moving out of the surface plane, the recorded intensity should drop on both channels simultaneously. Note that, in contrast to fluorescence from quantum dots or dyes, the total light scattering intensity stays constant over the entire length of the measurement (and indeed for many hours), without blinking or photobleaching. The scatting intensity Isca of a gold nanorod in one particular polarization direction is proportional to the square of the cosine of the angle θ between the rod and the polarization direction, assuming a homogeneous illumination. We can therefore deduce the orientation angle backward from the measurement by θ ) arccos(xIsca). The result is shown in the lower graph in Figure 2. The resulting angles are equally probable for long time traces for freely rotating particles, confirming the randomness of the observed motion. There are considerable variations between different particles, even in the same measurement. Representative examples are shown in Figure 3. Some particles show an angle distribution over time consistent with simple random twodimensional rotations on the 10 ms time scale (particles C and D). Others show long periods of no apparent orientation changes, or “sticky periods” in the second time scale (A and B). We believe the particles get trapped on the surface in a shallow enough potential to allow the particles eventually to escape and resume their rotational motion. It is currently not clear if this potential is due to surface roughness or chemical interactions. The fastest drops in autocorrelation times correspond to time constants of about 60 ms. The free rotational diffusion constant DR of our rods around their long axes is given by:25 DR )
3kBT πηl3
(ln2ld - 0.8) ≈ 2 × 10 s
4 -1
(η ) 1 mPas, l ) 60 nm, d ) 20 nm, kBT ) 4 × 10-21 J), which would mean an autocorrelation decay time constant of 1/6DR ≈10 µs. The considerably larger measured value Nano Lett., Vol. 5, No. 2, 2005
Figure 4. (a) Histogram of particles showing a given degree of rotational freedom Frot as defined in the text. The distribution of Frot shows an exponential decay toward larger values, which means most particles are very “sticky”. (b) The fraction of rotation times Frot for 79 particles (from one sample) derived from the first half of the experiment plotted against the second half of the experiment. Most points are relatively close to the y ) x line. This means particles do not change their individual “stickiness” or rotational freedom during our observation time (168 s), either because the observation time is too short or because there are intrinsic differences within the particle sample.
indicates that the rotational motion is dominated by the surface attachment and detachment, not by the viscosity of solvent. A large collection of time traces (1015 particles) collected with various frame rates shows a multitude of possible behaviors, from “freely rotating”, frequent short “sticky periods” to very long “sticky periods”. We take as measure of the rotational freedom the standard deviation over 60 ms and define values of greater than 12% of the mean value as “freely rotating”. The fraction of time particles that are “freely rotating” (Frot) indicates whether a particle is more “sticky” or “freely rotating”. The distribution of Frot over particles in the same sample drops off approximately exponentially to higher percentages (Figure 4a). The value of Frot for each particle does not change significantly from the first half of the experimental observation time to the second half (Figure 4b). This means that either the switching between sticky states and freely rotating states occurs on time scales greater than the maximal observation time (168 s) or 303
particles show different affinities to the surface, e.g., due to a different particle charge. The observed behavior is consistent with the following model: the particles slide randomly on the surface and the surface affinity varies between particles. The slow lateral diffusion of the rods remains unresolved by the microscope, limited by diffraction and the camera pixel size. The rotational motion, however, is readily observed by our polarization method, providing a probe for this otherwise hidden mechanism, which resembles the Le´vi flights observed for atoms and clusters on surfaces.26-28 In conclusion, we show that the polarized light scattering of gold nanorods may be used to monitor orientations on the nanoscale. By recording orthogonal polarizations simultaneously we show their two-dimensional confinement to the glass surface under the employed experimental conditions. The fastest rotational diffusion times are on the order of 60 ms, showing the large effective local viscosity near the surface dominated by the particle-surface interaction. Some particles also show extended “sticky times”, probably due to temporal attachment to the surface. The experimental setup is compatible with conventional single molecule setups for biomolecules. The light stability and high signal-to-noise ratio even with white light illumination from a tungsten lamp make our new method a very promising tool for the study of rotational motion in biomolecules and for the study of local surface properties. Acknowledgment. We acknowledge stimulating discussions and experimental help from Dr. Thorsten Hugel, Department of Physics, and Prof. Phillip Geissler, Department of Chemistry. This work was supported by NIH National Center for Research Resources through the University of California, Los Angeles, subaward agreement 0980GFD623, the U.S. Department of Energy under Contract No. DE-AC03-76SF00098, and by the Alexander von Humboldt Foundation through a Feodor Lynen Research Fellowship (C.S.).
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Supporting Information Available: Movie of intensity beating between the orthogonal polarization directions (frame rate 112 Hz). This material is available free of charge via the Internet at http://pubs.acs.org. References (1) Kulzer, F.; Orrit, M. Annu. ReV. Phys. Chem. 2004, 55, 585. (2) Prummer, M.; Sick, B.; Hecht, B.; Wild, U. P. J. Chem. Phys. 2003, 118, 9824. (3) Vacha, M.; Kotani, M. J. Chem. Phys. 2003, 118, 5279. (4) Azoulay, J.; Debarre, A.; Jaffiol, R.; Tchenio, P. Single Molecules 2001, 2, 241. (5) Weston, K. D.; Goldner, L. S. J. Phys. Chem. B 2001, 105, 3453. (6) Bartko, A. P.; Dickson, R. M. J. Phys. Chem. B 1999, 103, 11237. (7) Ha, T.; Laurence, T. A.; Chemla, D. S.; Weiss, S. J. Phys. Chem. B 1999, 103, 6839. (8) Sick, B.; Hecht, B.; Novotny, L. Phys. ReV. Lett. 2000, 85, 4482. (9) Bohmer, M.; Enderlein, J. JOSA B 2003, 20, 554. (10) Chung, I. H.; Shimizu, K. T.; Bawendi, M. G. Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 405. (11) Bryant, Z.; Stone, M. D.; Gore, J.; Smith, S. B.; Cozzarelli, N. R.; Bustamante, C. Nature 2003, 424, 338. (12) Yasuda, R.; Noji, H.; Yoshida, M.; Kinosita, K.; Itoh, H. Nature 2001, 410, 898. (13) Shelby, J. P.; Chiu, D. T. Lab on a Chip 2004, 4, 168. (14) Higgins, D. A.; Luther, B. J. J. Chem. Phys. 2003, 119, 3935. (15) VandenBout, D. A.; Kerimo, J.; Higgins, D. A.; Barbara, P. F. J. Phys. Chem. 1996, 100, 11843. (16) Noji, H.; Yasuda, R.; Yoshida, M.; Kinosita, K. Nature 1997, 386, 299. (17) Yguerabide, J.; Yguerabide, E. E. Anal. Biochem. 1998, 262, 157. (18) Schultz, D. A. Curr. Opin. Biotechnol. 2003, 14, 13. (19) Schultz, S.; Smith, D. R.; Mock, J. J.; Schultz, D. A. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 996. (20) So¨nnichsen, C.; Franzl, T.; Wilk, T.; von Plessen, G.; Feldmann, J.; Wilson, O.; Mulvaney, P. Phys. ReV. Lett. 2002, 88. (21) So¨nnichsen, C.; Geier, S.; Hecker, N. E.; von Plessen, G.; 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. (22) Boyer, D.; Tamarat, P.; Maali, A.; Lounis, B.; Orrit, M. Science 2002, 297, 1160. (23) So¨nnichsen, C. Plasmons in metal nanostructures; Cuvillier Verlag: Go¨ttingen, Germany, 2001. (24) Jana, N. R.; Gearheart, L.; Murphy, C. J. AdV. Mater. 2001, 13, 1389. (25) Li, L. S.; Alivisatos, A. P. Phys. ReV. Lett. 2003, 90. (26) Luedtke, W. D.; Landman, U. Phys. ReV. Lett. 1999, 82, 3835. (27) Naumovets, A. G.; Zhang, Z. Y. Surf. Sci. 2002, 500, 414. (28) Jensen, P.; Blase, X.; Ordejon, P. Surf. Sci. 2004, 564, 173.
NL048089K
Nano Lett., Vol. 5, No. 2, 2005
