Technical Note pubs.acs.org/ac
Subdiffraction-Limited Plasmonic Imaging with Anisotropic Metal Nanoparticles Xiaodong Cheng, Dinggui Dai, Dong Xu, Yan He,* and Edward S. Yeung State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, College of Biology, Hunan University, Changsha 410082, P. R. China S Supporting Information *
ABSTRACT: We have developed a high-resolution nonfluorescent imaging method based on superlocalization of gold nanorods (AuNRs). By taking advantage of their anisotropic optical property of the plasmonic scattering of AuNRs, selective imaging of only a fraction of AuNRs can be achieved by rotating the sample relative to the linear polarized illumination under cross-polarization microscopy with a high NA objective. The AuNR positions obtained from a series of images could then be used to reconstruct the overall image. Two AuNRs with center-to-center distances of 80 nm were successfully resolved. This simple but deterministic super-resolution imaging technique can potentially be used to fingerprint optically anisotropic metal nanoparticles and their assemblies for labeling, sensing, and encryption applications.
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reported,23,24 super-resolution imaging of neighboring plasmonic MNPs has been accomplished so far only via deconvolution of their LSPR spectra.25,26 However, emitters’ photoinstability is not the only physical property that can be exploited to realize optical state switching required for selective imaging. In this study, we report subdiffraction-limited MNP imaging with orientation-dependent localization microscopy (ODLM). By taking advantage of the polarization-sensitive property of gold nanorods (AuNRs), selective imaging of the anisotropic-shaped MNPs was achieved via changing the polarization of the incident beam relative to the orientation of the AuNRs. We show that a pair of AuNRs separated by as close as 80/30 nm center-to-center/edge-to-edge was successfully resolved. This simple technique could be utilized not only for super-resolution imaging of noncoupled MNPs but also to reveal detailed coupling modes of MNP nanostructures for various applications.
ith more and more studies in physical, biological, and material science being focused on complex systems at molecular and nanometer scales, there is an increasing demand on advanced imaging techniques that can detect and resolve nanometer-sized objects at spatial resolution below the optical diffraction limit. In recent years, by taking advantage of the photophysics of fluorescent emitters to selectively switch on/off close-by molecules or nanoparticles (NPs), functional superresolution microscopy such as stochastic optical reconstruction microscopy (STORM)1,2 and stimulated emission depletion fluorescence microscopy (STED)3,4 have been developed, which have pushed the resolution of fluorescence imaging down to 10−60 nm and have been utilized to study enzyme activities and the organizations of subcellular structures.5−8 But so far, similar selective imaging techniques that can probe the distributions and activities of nonfluorescent NPs at subdiffraction-limited resolution have not been reported. Due to their large cross sections, photostability and mild surface chemistry, noble metal nanoparticles (MNPs) that support localized surface plasmon resonance (LSPR) have emerged as an important class of nonfluorescence contrast agent.9−11 Plasmonic imaging of the distribution, assembly, and dynamics of MNPs has been used extensively to investigate MNP-based disease diagnosis,12,13 drug delivery,14,15 sensing,16,17 catalysis,18,19 photonic devices,20 etc. But different from fluorescent emitters, MNPs are optically stable and do not photobleach or photoblink under continuous illumination. While such high photostability facilitates their long-time observations, selective imaging of MNPs closely spaced within the diffraction limit cannot be achieved by temporally or spatially manipulating the intensity profile of the illumination light. Although a number of techniques are capable of superlocalization of single MNPs21,22 and MNP-assisted super-resolution of small dye molecules have also been © 2014 American Chemical Society
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EXPERIMENTAL SECTION Chemicals. Cetyl trimethylammonium bromide (CTAB), ammonium bromide (NH4Br), hydrogen peroxide (H2O2, 30%), HAuCl4·3H2O, ascorbic acid (AA), NaBH4, and AgNO3 were purchased from Sinopharm Chemical (Shanghai, China). AuNR Preparation. The seed-mediated growth method to fabricate CTAB-coated AuNRs has been described previously in the literature.27 Briefly, 4 mL of 0.1 M CTAB was gently mixed with 40.5 μL of 24.28 mM HAuCl4, and then 24 μL of 0.1 M ice-cold NaBH4 were rapidly added into the solution, followed by rapid mixing for 2 min. After the color of the AuNP Received: October 30, 2013 Accepted: February 6, 2014 Published: February 18, 2014 2303
dx.doi.org/10.1021/ac403512w | Anal. Chem. 2014, 86, 2303−2307
Analytical Chemistry
Technical Note
Figure 1. (a) Schematics of the optical setup of ODLM. (b) Illustration of ODLM imaging sequence. (c−e) Images of a group of randomly oriented AuNRs obtained by (c) DFM, (d) ODLM images at 6 different angles of the sample substrate, and (e) a reconstructed sum image from d.
consists of a polarizer, a NA = 1.40 oil immersion condenser, a NA = 1.40 100× Plan Apo VC oil immersion objective, and an analyzer. For dark field microscopy, a NA = 1.20−1.43 oil immersion darkfield condenser, and a 60× Plan Fluor oil immersion objective were used. The acquired images were analyzed using ImageJ. Data Analysis. The two-dimensional Gaussian model for fitting the center of the AuNR is given by the following equation,28,29
seed solution turned into pale brown-yellow, it was stored in a water bath maintained at 29 °C for 2 h before use. For AuNR preparation, 10 mL of 0.1 M CTAB and 206 μL of 24.28 mM HAuCl4 were gently mixed. Ten microliters of 0.1 M AgNO3 and 52.5 μL of 0.1 M AA were then injected into the mixture in order. After the solution turned colorless, 30 μL of AuNP seed solution was added into the growth solution, followed by rapid shaking of the test tube for 10 s, and left undisturbed for 3 h. After that, 1.5 mL of freshly prepared seed AuNR solution was quickly added into the mixture solution containing 10 mL of 0.05 M CTAB, 240 μL of 24.28 mM HAuCl4, and 93 μL of 0.1 M AA, followed by vigorous inversion for 5 s. The reaction solution was left undisturbed again for 20 min. The excess reagents were removed by washing with DI water 3 times. Morphology characterization of AuNRs was performed using transmission electron microscopy (TEM) (JEM1230, JEOL). All extinction spectra were measured using a USB2000+ microspectrometer from Ocean Optics. The as-prepared AuNRs have a LSPR spectral maximum at 650 nm with a mean width of 35 ± 6 nm and a mean length of 70 ± 9 nm (Figure S1 of the Supporting Information). Single Particle Experiment. In a typical experiment, 10 μL of diluted AuNR solution was dropped onto a copper grid and TEM images were taken after the droplet was dried. The copper grid was then immediately placed between a microscope slide and a coverglass. For sample protection, fingernail polish (refractive index of ∼1.42) was used to fill the empty space as the immersion medium. Optical Imaging. All optical imaging experiments were performed on a Nikon 80i microscope equipped with a 100 W halogen tungsten lamp and an Olympus DP72 color CCD camera. For cross-polarization microscopy, the light path
⎡ ⎛ (y − y0 )2 ⎞⎤ (x − x0)2 ⎟⎥ + I(x , y) = c + I0exp⎢ −⎜⎜ ⎢⎣ ⎝ 2wx 2 2wy 2 ⎟⎠⎥⎦
where x0 and y0 represent the position of the center of the Gaussian, I(x, y) and I0 are the peak intensity at pixel (x, y) and (x0, y0), wx and wy are the width of the Gaussian distribution, and c is the background.
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RESULTS AND DISCUSSION The basic principle of ODLM, as its name appears, is similar in form to photo-activated localization microscopy (PALM).30 But rather than stochastic, sequential activation of photoswitchable fluorescent molecules, ODLM works by deterministic, rotational illumination of polarization-sensitive plasmonic AuNRs. Thanks to the morphological asymmetry, the surface plasmon resonance of the rod-shaped MNPs is split into transverse and longitudinal modes.31 A reduction of plasmon damping in the latter gives rise to more efficient resonance light-scattering polarized parallel to the long axis, allowing AuNRs to be approximated to oscillating-dipole-like, polarization-sensitive emitters.32,33 When examined under crosspolarization microscopy (CPM) with the polarizer and the 2304
dx.doi.org/10.1021/ac403512w | Anal. Chem. 2014, 86, 2303−2307
Analytical Chemistry
Technical Note
Figure 2. (a) TEM (the scale bar is 1 μm) and (b) DFM images of AuNRs dispersed on a copper grid. The dash rectangle marked 4 differently orientated AuNRs. (c) Selective imaging (upper) and the corresponding contour plots (lower) of the 4 AuNRs at different angles. The scale bar is 500 nm. (d) The reconstructed contour plots from c.
having different azimuth angles (Figure 2a). From the TEM image, their center-to-center distances were measured to be D12 = 246 nm, D23 = 444 nm, and D34 = 307 nm. Under DFM (Figure 2b), neither P1 and P2 nor P3 and P4 can be resolved, and the 4 particles appear as 2 large spots. But with ODLM, we were able to selectively observe just P1 and P4, P2 and P4, or P2 and P3 at 3 different Ap. Two-dimensional (2D) Gaussian fitting of the intensity profile determined the centroid of each AuNR.28,29,35 Repetitive measurements after correction for sample drifting indicated that the localization precision was ∼15 nm. After image reconstruction through overlaying center positions of the same particles, D12, D23, and D34 were calculated to be 273, 449, and 283 nm, respectively, which closely matched the TEM results. Since there is no photobleaching and an infinite number of photons can be collected from a single MNP, the precision of MNP localization as well as interparticle distance measurement should, in principle, be less than 1 nm.36 We attribute the observed larger deviation to asymmetric LSPR intensity distribution profile of anisotropic objects.37 A localization error of
