Article pubs.acs.org/JPCC
Hyperspectral Dark Field Optical Microscopy of Single Silver Nanospheres Patrick Z. El-Khoury,* Alan G. Joly, and Wayne P. Hess* Physical Sciences Division, Pacific Northwest National Laboratory, P.O. Box 999, Richland, Washington 99352, United States ABSTRACT: We record spectrally (400 ≤ λ ≤ 675 nm, Δλ < 4.69 nm) and spatially (diffraction-limited, sampled at 85 nm2/ pixel) resolved dark field (DF) scattering from single silver nanospheres of 100 nm in diameter. Hyperspectral DF optical microscopy is achieved by coupling a hyperspectral detector to an optical microscope, whereby spectrally resolved diffractionlimited images of hundreds of silver nanoparticles can be recorded in ∼30 s. We demonstrate how the centers and edges of individual particles can be localized in two dimensions to within a single pixel (85 nm2), using a statistical method for examining texture based on a co-occurrence matrix. Subsequently, spatial averaging of the spectral response in a 3 × 3 pixel area around the particle centers affords ample signal to noise to resolve the plasmon resonance of a single silver nanosphere. A close inspection of the scattering spectra of 31 different nanospheres reveals that each particle has its unique (i) relative scattering efficiency and (ii) plasmon resonance maximum and dephasing time. These observations are suggestive of nanometric structural variations over length scales much finer than the spatial resolution attainable using the all-optical technique described herein.
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INTRODUCTION That many physical, chemical, and biological processes can be better understood from single-particle studies is a premise that has motivated significant advances in the field of plasmonics. This concept has not only steered the field toward singlemolecule chemical detection,1,2 imaging,3 and dynamics,4 but has also driven technological developments aimed at characterizing the properties of individual plasmonic nanostructures.5 In this regard, by virtue of their polaritonic nature,6 both photons and electrons may be used to probe the plasmonic Eigenmodes of nanostructured noble metals. As such, several far field and near field techniques powered by photons, electrons, and photoelectrons have been developed and/or adopted to probe various manifestations of the plasmonic response in singlemetal nanoparticles, as illustrated in a recent review.5 Of the various far field techniques used to access the plasmonic properties of single nanometric scatterers, the inherent simplicity and rich information content in dark field (DF) optical microscopy and spectroscopy is attractive.5,7−10 Herein, we illustrate that by coupling a hyperspectral detector equipped with an internal scanning design to a standard optical microscope capable of DF imaging, three-dimensional (3D) (x, y, λ) spectral images of hundreds of plasmonic nanoparticles can be acquired in ∼30 s. We note that although this work is focused on hyperspectral DF optical microscopy, spectrally resolved transmission, reflection, and fluorescence optical micrographs can be readily recorded using the setup described herein; the target systems that can be potentially addressed using this all-optical technique span the realms of several disciplines. Notably, our approach requires neither sample scanning nor point-by-point detection schemes, which (i) are © XXXX American Chemical Society
typically more time-consuming, (ii) may introduce uncertainties (e.g., as a result of image drifting over time), and (iii) often involve more convoluted setups. In the following, we discuss hyperspectral DF optical microscopy measurements and their analysis, through an exemplary study targeting plasmonic silver nanospheres of 100 nm in diameter.
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METHODS Our hyperspectral optical microscope consists of a hyperspectral detector (SOC710-VP, Surface Optics Corporation, San Diego, CA) equipped with an internal scanner coupled to a confocal optical microscope (DM4000M, Leica, Wetzlar, Germany). The setup currently allows us to record spectrally (400 ≤ λ ≤ 1000 nm, Δλ < 4.69 nm) and spatially (diffractionlimited, sampled at 85 nm2/pixel) resolved optical micrographs by scanning a line detector (at a rate of 30 lines/s) containing 512 pixels (85 nm2/pixel) over the field of view dictated by the microscope objective (Leica N PLAN L 100x/0.75 BD). Each element comprising the aforementioned line detector contains spectral information in the 400−1000 nm region. In this work, the hyperspectral images of the sample were spatially and spectrally normalized to a reference hyperspectral image collected from the light source (depolarized/incoherent), incident onto a diffusive reflection standard (Ocean Optics, WS-1-SL). The recorded images were analyzed using the ENVI 5.1 (Exelis Visual Information Solutions). The same software Received: March 7, 2016 Revised: March 14, 2016
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DOI: 10.1021/acs.jpcc.6b02401 J. Phys. Chem. C XXXX, XXX, XXX−XXX
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The Journal of Physical Chemistry C
of the microscope objective used herein (100×, NA = 0.75). Each pixel comprising the aforementioned line detector contains spectral information in the 400−675 nm spectral region. The resulting hyperspectral data cube can be analyzed by taking image slices at different wavelengths as shown in Figure 2B, and/or by inspecting the spectra contained in each pixel, vide infra. A selected region of the 3D hyperspectral DF image that contains a single silver nanoparticle constitutes the starting point of our discussion. Single-Particle Spatial Analysis. Figure 3A shows the 503 nm DF optical image of an isolated silver nanoparticle, a horizontal cross section of which is plotted in Figure 3C. A Gaussian fit of the recorded scattering profile indicated a full width at half-maximum of 381 nm. This is consistent with, but somewhat larger than, the diffraction-limited ∼335 nm value expected based on a numerical aperture of 0.75. The experimental value depends on the choice of physical model used and overall quality of the resulting fit. For instance, the deviation of the Gaussian fit from the experimental profile toward the maximum is noted; see Figure 3C. Alternatively, we may analyze the scattering profile of the isolated nanoparticle using a statistical method which solely relies on intensity counts to examine image texture. Herein, we opt to apply a texture filter based on a co-occurrence matrix.11 Our filter of choice for this study is statistical variance in a 3 × 3 pixel window, which is scanned across the recorded image.11 This allows us to rapidly locate the center and edges (in the diffraction-limited far field image projection) of the isolated scatterer from its hyperspectral DF image. Namely, applying a variance filter to the DF image shown in Figure 3A yields the filtered image shown in Figure 3B. The 2D scattering profile in the former, best described as a 2D normal distribution, is transformed into a doughnut-shaped spatial distribution in the latter. This is a consequence of low local variance at the center of the scattering profile, as compared to high local variance toward its edges, or more accurately, at the inflection points of the 2D normal intensity distribution. The concept is further bolstered in Figure 3C, where a horizontal cross section of the variance-filtered image is plotted. Here, the scattering diameter measured from the variance-filtered image as the distance between the two points of highest variance is 340 nm, in close agreement with the theoretical value of ∼335 nm based on the numerical aperture of our microscope objective. Applying a variance filter based on a co-occurrence matrix also allows us to rapidly pinpoint the center of the scatterer in 2D, in this case to within one pixel (85 nm2); see Figure 3B,C. Subsequently, the spectra recorded in a 3 × 3 pixel area around the identified particle center can be extracted from the hyperspectral optical image and averaged, as discussed below. Multiple (Single) Particle Spectral Analysis. The signal to noise achieved in the spatially averaged scattering spectrum enables us to record the optical response of a single plasmonic silver nanoparticle (see Figure 3D), which in this case is governed by the surface plasmon resonance of a spherical silver nanoparticle. Repeating the above-described single-nanoparticle analysis procedure for 31 different nanoparticles in our field of view allows us to readily resolve their individual plasmon resonances; see Figure 4. We observe marked differences between the optical signatures of the different isolated plasmonic nanoparticles in terms of (i) their relative scattering efficiencies, (ii) their resonance maxima, and (iii) their derived peak widths. The first observation is consistent with a recent report from our group,12 where correlated scanning electron
was used to perform statistical analysis based on co-occurrence measures. The sample was prepared by drop casting a solution of citrate-stabilized silver nanoparticles (Sigma-Aldrich, St. Louis, diameter = 100 nm) on a standard glass microscope slide.
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RESULTS AND DISCUSSION Measurement. With use of an optical microscope operating in DF mode, a real color image of the scattering from an isolated silver nanoparticle can be recorded; see Figure 1A. This
Figure 1. Panel A shows a real color image of the scattering from an isolated silver nanoparticle (diameter = 100 nm) on glass. Panel B schematically illustrates the underlying concept behind hyperspectral optical microscopy.
can be achieved using any standard microscope camera. Our goal is to spectrally resolve the aforementioned DF optical image, as schematically illustrated in Figure 1B, and subsequently, to measure the plasmon resonance of the isolated silver nanoparticle imaged in Figure 1A. This is achieved by coupling a hyperspectral detector to a confocal optical microscope. Figure 2A shows a 3D representation of a hyperspectral DF optical image of a sparse distribution of 100 nm silver nanoparticles on a glass microscope slide. This image contains both spatial and spectral information, obtained by scanning a line detector containing 512 pixels (85 nm2/ pixel) over the field of view dictated by the numerical aperture
Figure 2. Panel A shows a 3D representation of a hyperspectral dark field micrograph of 100 nm silver nanospheres dispersed on a glass microscope slide. The field of view in this panel is ∼44 × 59 μm2. Three regions are highlighted in this image. The first is a solid white rectangle, illustrating the field of view shown in panel B. The second is a solid red square highlighting a single particle, a detailed analysis of which is performed as shown in Figure 3. The third is a dashed white rectangle depicting an area of interest further analyzed in Figure 4. (B) Wavelength-resolved dark field optical images (slices from panel A) at the wavelengths indicated in the inset. B
DOI: 10.1021/acs.jpcc.6b02401 J. Phys. Chem. C XXXX, XXX, XXX−XXX
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The Journal of Physical Chemistry C
Figure 3. Panel A shows the 503 nm dark field optical image of a single particle, which after applying a variance filter based on a co-occurrence matrix (3 × 3 pixel window) yields the image shown in panel B. In panel C, horizontal cross sections taken from panels A and B are plotted. Shown in the same panel is a Gaussian fit to the scattering cross section taken from panel A. Panel D illustrates nine dark field scattering spectra extracted from individual pixels contained in the 3 × 3 pixel area highlighted using a solid black square in panel A. The thick red line represents the average of all nine spectra, namely, the average scattering from an isolated silver nanosphere. The scale bars in panels A and B indicate 255 nm.
Figure 4. A selected area 503 nm dark field optical image is shown in panel A, which after applying a variance filter based on a co-occurrence matrix (3 × 3 pixel window) yields the image shown in panel B. A total of 31 particles are highlighted in panel A, and their spatially averaged spectra (in a 3 × 3 pixel area) are stacked in panel C. Seven different single-particle scattering spectra are highlighted using asterisks in panel C, and in turn shown on the same plot in panel D. Panel D also shows the averaged scattering from all 31 individual particles highlighted in panel A. Note that a single scaling factor was used for all of the spectra plotted in panels C and D; the relative intensities correspond to different scattering efficiencies of the different particles considered in this analysis.
are 1.9 and 2.7 fs, respectively. As no obvious correlations were observed between the scattering efficiencies, plasmon resonance maxima, and the derived plasmon dephasing times of the individual particles, the overall observed differences between the optical signatures of the individual particles are attributed to the nanometric structural specifics of each of the particles probed.12 Further work is warranted in this regard; correlating the recorded hyperspectral images to topographic scanning electron/helium ion/transmission electron micrographs is a challenge we take on in future works.
and nonlinear photoemissions electron micrographs of plasmonic silver nanoparticles revealed that nanometric structural defects govern the magnitudes (and 2D spatial profiles) of the surface plasmon-enhanced local electric fields. The second finding is also somewhat intuitive; contributions from individual nanoparticles with slightly different plasmon resonances sum up to an overall broadened line shape in typical ensemble averaged (e.g., colloidal) measurements. The third observation is more informative; see Figure 4C,D. Namely, the line width of the measured resonances can be directly related to the dephasing of the coherent electron oscillations.13 Because of the non-Lorentzian profile of the scattering cross section, the band width typically used to define the plasmon dephasing time is the red half width of the absorption band, as T2 = ℏ/Γ1/2.13 We find that the average plasmon dephasing time (for all 31 particles considered in this analysis) is ∼2.4 fs, similar to previously reported values13 for plasmonic gold nanoparticles featuring comparable diameters. As evident from Figure 4D, the averaged value masks a spread in dephasing times obtained from the different single particles probed herein. For instance, the plasmon dephasing time calculated for particles 1 and 20
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CONCLUSIONS We record hyperspectral DF optical images of 100 nm plasmonic silver nanospheres. Hyperspectral optical microscopy is achieved by coupling a hyperspectral detector to a standard optical microscope, allowing us to record diffraction-limited spectral images of hundreds of nanospheres in our field of view. We analyze both the spatial and spectral response of individual silver nanospheres. Ample signal to noise is achieved; we resolve the plasmon resonance and extract the plasmon dephasing time of individual silver nanoparticles. A close C
DOI: 10.1021/acs.jpcc.6b02401 J. Phys. Chem. C XXXX, XXX, XXX−XXX
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(4) Yampolsky, S.; Fishman, D. A.; Dey, S.; Hulkko, E.; Banik, M.; Potma, E. O.; Apkarian, V. A. Seeing a Single Molecule Vibrate through Time-Resolved Coherent Anti-Stokes Raman Scattering. Nat. Photonics 2014, 8, 650−656. (5) Ringe, E.; Sharma, B.; Henry, A.; Marks, L. D.; Van Duyne, R. P. Single Nanoparticle Plasmonics. Phys. Chem. Chem. Phys. 2013, 15, 4110−4129. (6) Pitarke, J. M.; Silkin, V. M.; Chulkov, E. V.; Echenique, P. M. Theory of Surface Plasmons and Surface-Plasmon Polaritons. Rep. Prog. Phys. 2007, 70, 1−87. (7) Chang, W. S.; Slaughter, L. S.; Khanal, B. P.; Manna, P.; Zubarev, E. R.; Link, S. One-Dimensional Coupling of Gold Nanoparticle Plasmons in Self-Assembled Ring Superstructures. Nano Lett. 2009, 9, 1152−1157. (8) Lindfors, K.; Kalkbrenner, T.; Stoller, P.; Sandoghdar, V. Detection and Spectroscopy of Gold Nanoparticles Using Supercontinuum White Light Confocal Microscopy. Phys. Rev. Lett. 2004, 93, 037401. (9) Novo, C.; Gomez, D.; Perez-Juste, J.; Zhang, Z.; Petrova, H.; Reismann, M.; Mulvaney, P.; Hartland, G. V. Contributions from Radiation Samping and Surface Scattering to the Linewidth of the Longitudinal Plasmon Band of Gold Nanorods: a Single Particle Study. Phys. Chem. Chem. Phys. 2006, 8, 3540−3546. (10) Tcherniak, A.; Ha, J. W.; Dominguez-Medina, S.; Slaughter, L. S.; Link, S. Probing a Century Old Prediction One Plasmonic Particle at a Time. Nano Lett. 2010, 10, 1398−1404. (11) Haralick, R.; Shanmugam, K.; Dinstein, I. Textural Features for Image Classification. IEEE Trans. Syst., Man, and Cybern. 1973, 3 (6), 610−621. (12) Peppernick, S. J.; Joly, A. G.; Beck, K. M.; Hess, W. P. PlasmonInduced Optical Field Enhancement Studied by Correlated Scanning and Photoemission Electron Microscopy. J. Chem. Phys. 2013, 138, 154701. (13) Link, S.; El Sayed, M. A. Size and Temperature Dependence of the Plasmon Absorption of Colloidal Gold Nanoparticles. J. Phys. Chem. B 1999, 103, 4212−4217.
inspection of the scattering spectra of 31 different nanospheres reveals that each particle has its unique character, attributed to nanometric structural variations over length scales much finer than the spatial resolution attainable using the all-optical technique described herein. Beyond the realm of plasmonic metal nanospheres, numerous potential applications of the hyperspectral optical microscope described in this work are envisioned. Coupling the hyperspectral detector to a surfaceenhanced Raman scattering (SERS) microscope where chemical maps can be simultaneously recoded is a logical extension to this work. The ability to locate and measure the optical response of single plasmonic particles (and their assemblies) to which molecular reporters are chemisorbed will allow us to further optimize SERS (as well tip-enhanced Raman scattering, TERS) experiments. For instance, given a laser excitation wavelength of 633 nm, the SERS response of molecular reporters adsorbed onto particle 1 would be expected to be brighter than the optical response of molecules in the vicinity of particle 20; see Figure 4D. Collecting hyperspectral DF images of SERS/TERS constructs prior to SERS/TERS measurements is invaluable in this regard. 4D (x, y, z, λ) optical microscopy in transmission, reflection, fluorescence, and DF modes is another possibility that would only require sample translation in one dimension, namely, along the z-direction of light propagation. Both of the aforementioned developments will be detailed in follow-up works.
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AUTHOR INFORMATION
Corresponding Authors
*Pacific Northwest National Laboratory, 902 Batelle Boulevard, P.O. Box 999, MSIN K8-88, Richland, WA 99352, USA. E-mail:
[email protected]. Tel.: 509-371-6048. Fax: 509-3716145. *E-mail:
[email protected]. Tel.: 509-371-6140. Fax: 509371-6145. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS P.Z.E. acknowledges partial support from the Laboratory Directed Research and Development Program through a Linus Pauling Fellowship at Pacific Northwest National Laboratory (PNNL). The authors acknowledge support from the U.S. Department of Energy (DOE), Office of Science, Office of Basic Energy Sciences, Division of Chemical Sciences, Geosciences & Biosciences. This work was performed in EMSL, a national scientific user facility sponsored by DOE’s Office of Biological and Environmental Research and located at PNNL. PNNL is operated by Battelle Memorial Institute for the United States Department of Energy under DOE contract number DE-AC05-76RL1830.
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REFERENCES
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DOI: 10.1021/acs.jpcc.6b02401 J. Phys. Chem. C XXXX, XXX, XXX−XXX
