Letter pubs.acs.org/journal/apchd5
Wavelength-Dependent Super-resolution Images of Dye Molecules Coupled to Plasmonic Nanotriangles Esther A. Wertz,† Benjamin P. Isaacoff, and Julie S. Biteen* Department of Chemistry, University of Michigan, Ann Arbor, Michigan 48109-1055, United States S Supporting Information *
ABSTRACT: The emission properties of fluorescent molecules are strongly affected by proximal plasmonic nanoparticles that act as optical nanoantennas. In particular, fluorescence intensity can be greatly increased by enhancing both the excitation and emission rates of a fluorophore, and the angular and spatial emission pattern from a dye coupled to a plasmonic nanoantenna will be altered. Here, we use single-molecule imaging to measure this shifted emission pattern based on the super-resolution image of cyanine dye molecules coupled to gold nanotriangles. To compare the relative effects of excitation versus emission enhancement on the emission pattern, we vary laser excitation wavelengths, dye emission and absorbance spectra, and local surface plasmon resonance frequency. We demonstrate that the emission pattern is dramatically changed when coupling occurs and that coupling between the dye and gold nanotriangle happens even in the absence of intensity enhancement. KEYWORDS: plasmon-enhanced emission, plasmon-enhanced excitation, nanoantenna, single-molecule microscopy, super-resolution imaging, light−matter interactions maximum in the fluorescence intensity,14 and this trade-off between enhancement and quenching has been measured for individual dye molecules by single-molecule fluorescence (SMF) detection.15 Furthermore, in addition to being distance-dependent, the enhancement strength is also strongly affected by the spectral overlap of the fluorescence excitation and emission with the LSPR spectrum.16−22 Although fluorescence modification by nanoparticles and the effects of the excitation wavelength and the emission spectrum on the emission of the coupled system have been extensively investigated experimentally and theoretically,8,11,16−22 very few studies have examined how the coupling affects the emission pattern on the nanoscale: the direction and position of the emission from a dye coupled to a plasmonic nanoantenna will be altered.23−27 Overall, any increase in fluorescence is the product of enhanced excitation and enhanced emission, yet the relative importance of these two sources of enhancement on the magnitude and spatial pattern of the emission is not well understood. Previous studies of plasmon-enhanced fluorescence have predominantly focused on maximizing fluorescence enhancement and have shown that this enhancement correlates with properties such as the antenna size, shape, and spectral response. Studies separating the respective effects of enhanced emission and enhanced excitation on the emission intensity have shown the importance of excitation wavelength17,22 as well
M
etal nanoparticles sustain a collective oscillation of their free electrons, called a localized surface plasmon resonance (LSPR), when excited by an electromagnetic (EM) wave. When this incident EM wave is resonant with the LSPR frequency, the EM field intensity is strongly increased in the near field of the nanoantenna.1,2 These resonances thus produce strong, localized EM fields, which have been used to improve devices such as biosensors and light-emitting devices and which enable spectroscopies such as surface-enhanced Raman scattering and plasmon-enhanced fluorescence.3−7 Overall, these applications rely on the ability of LSPRs to drastically modify the local environment of a nearby dipole emitter.8,9 In particular, a plasmonic nanoantenna will enhance fluorescence by concentrating incident light into a subdiffraction-limited volume,10 which leads to enhanced absorption (excitation enhancement) by a proximal dye, and by increasing the local density of optical states (LDOS) about the nanoparticle, which changes both the fluorescence lifetime and the quantum yield of the nearby fluorophore (emission enhancement).11 The enhancement processes can be controlled by tuning the LSPR properties, which vary with nanoparticle size, shape, and material. While the emission of a molecule directly on a metal surface will be quenched,12 the enhanced LDOS in the nanoparticle near field can strongly enhance the emission of a dye molecule a few nanometers away from a nanoparticle surface.8,11,13 Studies of the changes in measured fluorescence intensity as a function of the separation distance between a dipole emitter and a nanoparticle have demonstrated a local © 2016 American Chemical Society
Received: May 18, 2016 Published: September 20, 2016 1733
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Figure 1. Nanotriangle (NT) array characterization. Measured dark-field spectra of (a) 85 nm, (b) 130 nm, and (c) 145 nm side length NTs on ITO in water (black lines) and the corresponding calculated scattering cross sections (blue lines); the insets show corresponding SEM images. Scale bars: 100 nm. Normalized measured bulk absorption (red lines) and fluorescence emission (brown lines) spectra of (d) Cy3, (e) Cy5, and (f) Cy5.5. The emission spectrum of Cy3 was measured here with 532 nm excitation, Cy5 with 561 nm excitation, and Cy5.5 with 640 nm excitation. The different laser excitation lines used for each dye single-molecule experiment are also indicated (dashed lines).
as dye emission wavelength on the overall fluorescence enhancement factor.18−21 In this Letter, we measure nanometer-scale shifts in the emission pattern from dye molecules coupled to gold nanoparticles by using single-molecule imaging to detect the precise position of fluorescence emission. We consider three dyes of different colors excited with four different laser frequencies and coupled to a range of gold nanotriangles (NTs) with increasing sizesand therefore LSPR wavelengthsto investigate how the dependence of the fluorescence enhancement on excitation and emission wavelengths affects the emission pattern from the coupled dye−nanoparticle system using super-resolution imaging. To investigate the respective roles of plasmon-enhanced excitation and emission on the fluorescence properties of nearby dye molecules, we fabricated gold NT arrays by electron-beam lithography on an indium tin oxide (ITO)coated glass coverslip. Each of the eight arrays consisted of 50 nm thick NTs arranged with a 500 nm center-to-center pitch and a specific NT side length between 75 and 145 nm, such that each NT array was characterized by a distinct plasmon resonance frequency. The dark-field scattering spectra of NTs in three representative arrays are plotted in black in Figure 1a−c. As is seen in the scanning electron microscope images in the insets of Figure 1a−c, the NT tips are rounded. Because the NT sizes depend strongly on the lithography process and may differ slightly from the initial design, the true NT size for each array (Figure S1, Table S1) is therefore estimated by comparing the measured dark-field scattering spectra (black curves in Figure 1a−c) to the scattering spectra from a single rounded NT with the same sample geometry as calculated with full-field electromagnetic finite difference time-domain (FDTD) simulations (blue curves in Figure 1a−c; Methods). While the scattering simulations were performed on a single representative NT, the experimental dark-field spectra were obtained by integrating the signal over many particles within the array (several hundred NTs), which could result in broader line widths.28,29 The NTs were coupled to three different organic dyes (Cy3, Cy5, and Cy5.5), which gave us access to a range of fluorophore absorption and emission wavelengths. The measured absorption and emission spectra of these dyes in
solution are plotted in Figure 1d−f in red and brown, respectively. Finally, four different laser wavelengths were used to excite the NT−dye coupled system: 515, 532, 561, and 640 nm; these laser wavelengths are indicated by dashed black lines in Figure 1d−f. At the start of each experiment, the NT sample was immersed in a dilute solution of one of the cyanine dyes (Cy3, Cy5, or Cy5.5 in water), and at the end of the experiment, the sample was rinsed and then soaked overnight in acetone to avoid cross-contamination by any remaining dye molecules. Additionally, any molecules remaining on the surface were photobleached by exposing the sample to a higher powered laser beam before the start of each experiment (Methods). Single-molecule epifluorescence experiments were performed on each of the NT arrays with circularly polarized laser excitation using the PAINT (points accumulation for imaging in nanoscale topography)30 imaging technique (Methods).26 Briefly, with this technique, we image fluorescent molecules one at a time as they adsorb at random positions on the sample surface. While the free molecules diffuse too fast to be imaged on the EMCCD camera (integration time = 40 ms), the adsorbed molecules appear as bright punctate spots (Figure 2a,b). In the epifluorescence images, photoluminescence from the gold NTs produces a constant background, which is subsequently subtracted from all images taken in the same experimental conditions. To reduce the effect of variations in the NT scattering intensities, we systematically looked at the same corner of each array to ensure that all experiments for a given NT size were performed on the same particles. Figure 2a,b show a typical background-subtracted image of two Cy5.5 molecules adsorbing on an ITO-coated coverslip reference sample (Figure 2a) and near 130 nm NTs (LSPR resonance maximum = 720 nm; Figure 2b) on the same grayscale. The intensities of the NT-coupled molecules are brighter than the intensities of the reference molecules, illustrating a situation where plasmon enhancement is observed. The fluorescence intensity and apparent position of each detected dye molecule is then obtained from Gaussian fits to the background-subtracted SM emission profiles 31 and recorded for all detected molecules for each laser−dye−NT combination. The distribution of intensities for the reference 1734
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Figure 2. Wavelength-dependent plasmon-enhanced fluorescence. (a, b) Representative background-subtracted epifluorescence images of single Cy5.5 molecules adsorbed on a reference ITO substrate and near 130 nm NTs, respectively, on the same grayscale. Scale bars: 500 nm. (c) Normalized distribution of single-molecule fluorescence intensities of Cy5.5 molecules adsorbing on a reference ITO substrate (gray) and near 130 nm NTs (red); excitation wavelength = 640 nm. (d−h) Measured relative fluorescence intensities of all detected adsorbed single dye molecules (filled circles) and calculated average field intensity enhancement (open triangles) for different dyes and laser excitation wavelengths, as indicated on the panels. The colored arrows indicate the laser excitation wavelength. The intensity enhancement is defined as the ratio of the mean intensity from NT-coupled molecules to the mean intensity from molecules on the ITO reference, calculated from histograms like (c).
overlays the dye absorption and emission spectra with the measured maximum LSPR wavelength. If we were to simply consider the spectral overlap between the NT plasmon resonance peak and both the absorption and emission spectra in Figure S2, we would expect maximum enhancement to occur for Cy5 and Cy5.5 coupled to 120 or 130 nm NTs (LSPR resonance maximum = 660 and 725 nm, respectively). Our experiments indicate that under 640 nm excitation, both Cy5 and Cy5.5 show maximum fluorescence enhancement near this predicted point: when coupled to the 140 nm NTs (LSPR resonance maximum = 750 nm; Figure 2d,e). However, Cy5 excited with the 561 nm laser is maximally enhanced when coupled to the 100 nm NTs (LSPR resonance maximum = 625 nm; Figure 2f). Similarly, the Cy3 fluorescence enhancement is maximized for different NT sizes depending on the excitation laser wavelength. With 532 nm excitation, a clear maximum in the relative intensity is observed for the 100 and 120 nm NTs (LSPR resonance maximum = 625 and 660 nm, respectively; Figure 2g); this maximum does not overlap with the maximum dye absorption or emission wavelength due to the fact that the smaller particles are relatively poor scatterers. The scattering spectra of the larger particles, which scatter light much more effectively, overlap with the tail of the emission spectrum, and the molecules emitting at those higher wavelengths are thus more enhanced than those at lower wavelengths. With 515 nm excitation, very little Cy3 fluorescence enhancement is observed for any of the NT sizes, and no clear trend emerges in the relative fluorescence intensity (Figure 2h). Overall, in good agreement with previous studies,17,22 we observe that spectral overlap between the excitation and LSPR wavelengths is an important variable in determining the extent of plasmon enhancement in a dye−nanoparticle interaction. In the vicinity of a plasmonic nanoparticle, the fluorescence enhancement is proportional to the EM field enhancement.8,11,33 Indeed, FDTD simulations of the electric field intensity about an NT show that the amplitude of the EM field
molecules is then compared to the intensity distribution of the coupled molecules.32 Figure 2c shows the emission intensity distributions for Cy5.5 with 640 nm excitation, with and without coupling to 130 nm NTs. Cy5.5 molecules coupled to the NTs (red) show a significantly broader and brighter distribution of fluorescence intensities compared to the reference dye molecules (gray), indicating plasmon-enhanced fluorescence. Figure 2d−h summarize the fluorescence intensity results from all the experiments. Each black circle corresponds to the ratio of the average fluorescence intensity in the coupled dye− NT experiment to the average fluorescence intensity of the dye molecules on a reference ITO sample. For example, the 130 nm NT point (peak LSPR = 625 nm) in Figure 2d (fluorescence intensity enhancement = 2.5) was obtained from the data in Figure 2c by dividing the mean of the coupled (red) intensity distribution by the mean of the reference (gray) intensity distribution. Factors such as the dye quantum yield (QY) and the NT scattering cross section can strongly affect the emission enhancement strength.11,21,22 In particular, low-QY emitters can be more strongly enhanced than high-QY emitters, and this factor in conjunction with the fact that enhancement is proportional to the scattering cross section of the NT, plays an important role in whether the dye emission will be enhanced or quenched. However, if the same dye is used with different excitation lasers, there will be no change in the emission enhancement, while the excitation enhancement will be strongly affected by the change in absorption cross section of the NT at these different wavelengths. Furthermore, all of the cyanine dyes used in this study have intermediate QYs between 15% and 30%. Thus, the spectral overlap between dye and LSPR spectra is the critical parameter here for observing strong enhancement. Due to the Stokes shift between the absorption and emission spectrum (Figure 1d−f), the excitation laser wavelength must be considered as well.17,18,20 Figure S2 1735
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Figure 3. Super-resolution maps of apparent emission positions. Each dot shows the apparent emission position, relative to the closest NT position, of a single dye molecule adsorbed at a random position on the sample surface. (a) Cy5 on ITO reference with 640 nm excitation, (b−e) dye molecules near NTs with fluorescence excitation wavelength and NT sizes as indicated. Scale bars: 100 nm. Color bars: Fluorescence intensity enhancement. The white triangles in (b)−(f) indicate the NT position.
the area immediately adjacent to the NT, as well as on the NT. Experimentally, we observe no clear increase in the relative fluorescence enhancement only for Cy3 with 515 nm excitation (Figure 2h). In this case, the excitation wavelength is below the plasma frequency of gold and thus cannot excite the NT LSPRs. On the other hand, fluorescence enhancement is observed for 532 and 561 nm excitation of dyes in Figure 2f and h. Here, there is poor overlap of the laser wavelength with the LSPR, and the excitation enhancement is negligible; the increase in fluorescence intensity is mainly due to emission enhancement. Thus, in experiments with 640 nm excitation, the higher enhancement factors can be attributed to concurrent excitation and emission enhancement effects, while the lower excitation wavelengths (561 and 532 nm) allow us to measure the contribution of emission enhancement alone. The fluorescence emission of a dye molecule will be altered upon near-field coupling to a metal nanoparticle; this change will be reflected in a shift between the actual position and the apparent position detected in the far field. The PAINT superresolution imaging technique used here precisely determines the apparent position of single-molecule emission in addition to measuring the emission intensity.26 We can therefore map out how the coupled emission pattern changes with each dye molecule/laser excitation combination. Six representative maps are plotted in Figure 3, where each dot denotes the apparent emission position from a single molecule and the dot color corresponds to the amount of fluorescence intensity enhancement. For each panel, all the single-molecule emission measurements around approximately 100 NTs of the same size were collapsed onto a single relative coordinate to improve the statistics. In the reference case (Figure 3a), where the dyes adsorb at random positions on an ITO-coated coverslip with no NTs, the emission map shows a homogeneous distribution of dyes in space as well as a narrow intensity distribution. In
in the vicinity of an NT is strongly wavelength-dependent. The field intensity enhancement maps in Figure S3 are obtained by computing the field intensity, |E|2, at each excitation wavelength in the presence and absence of a gold NT and taking the ratio of these |E|2 values at each point in the plane of the NT. The average field enhancement is then calculated by taking the average of this enhancement map over the 500 nm × 500 nm area surrounding the NT and excluding the area directly on the triangle (where dyes would be strongly quenched and thus not detected). Using these field maps, we calculate for each laser excitation wavelength the average field intensity enhancementand thus the predicted excitation enhancementabout the NTs (blue triangles in Figure 2d−h) and compare this expected field intensity enhancement to the measured fluorescence intensity enhancement. For the case of 640 nm excitation (Figure 2d,e), the average field intensity enhancement calculation (blue triangles) is sufficient to qualitatively reproduce the experimental trends in fluorescence enhancement (black circles), indicating that in this case enhanced excitation is the main effect responsible for the changes in fluorescence intensity with NT size. However, this trend is not the case for the lower excitation wavelengths. In Figure 2f−h (laser wavelengths 561, 532, and 515 nm, respectively), the average field intensity is essentially unchanged for all NT sizes. These values of |E|2 < 1 in the simulation result from the fact that a finite amount of energy is input into the simulation. The plane wave amplitude is equal in the NT simulation and the reference simulation. The metal NT, which is highly absorptive, absorbs a large portion of that input energy. While all NTs absorb strongly, when the excitation is resonant with the LSPR, the concomitant reshaping of the incident field into the hot spots around the NT tends to eclipse these losses. This compensation is not the case off-resonance, where the only observed effect is a decreased field intensity in 1736
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Figure 4. Simulations of dipoles coupled to a nanoantenna. (a) Simulated dipole positions around an NT (blue dots), corresponding to the minimum symmetry unit of the triangle. At each position, two orthogonal dipole orientations were simulated, as indicated by the two arrows. The results for the blue dots were mapped to results for the red dots by symmetry, and these results were interpolated then randomly sampled to generate panels b−e. (b−e) Simulated apparent emission positions at three different wavelengths corresponding to the emission maxima of the cyanine dyes used in the experiments: (b) 700 nm/Cy5.5, (c, d) 670 nm/Cy5, and (e) 570 nm/Cy3. Scale bars: 100 nm. Color bars: Intensity of the simulated single-molecule emission. The black triangles indicate the NT position. (f) Simulated intensity enhancement vs mislocalization error magnitude for dipoles oriented along the x-axis (orange) or the y-axis (purple) at 670 nm near 130 nm NTs as in (c).
from the NT position, while the distribution of fit positions remains homogeneous farther from the NT (Figure 3f and Figure S4f). This behavior is completely opposite that of Cy3 with 532 nm excitation (Figure 3e and Figure S4e), in which case coupled molecules are shifted toward the NT center, showing again the importance of the laser excitation wavelength in the coupling between a dye and a nanoantenna. To better understand the coupled emission patterns, we simulated the interactions between a single dipole emitter and an NT in the same conditions as our experiments (Methods).26 To reproduce the experimental image of a nanoparticle-coupled single molecule, we focused the radiation distribution of a dipole near an NT onto the far-field imaging plane.27,34 This simulated diffraction-limited image was then processed in the same way as the experimental data: fit to a 2D Gaussian function to extract the apparent position and intensity. Two single dipole emitters (with dipole orientations in the x direction and the y direction, respectively) were placed one at a time at each position about the NT indicated by a blue dot in Figure 4a. The apparent emission positions of each dipole are plotted as dots in Figure 4b−e after randomly sampling interpolated results (Methods) to yield the expected distribution of molecule localizations at three different wavelengths corresponding to the emission maxima of the three dyes (700, 670, and 570 nm for Cy5.5, Cy5, and Cy3, respectively). These distributions are plotted for all NT sizes and all dye wavelengths in Figure S5. Although no dyes were positioned less than 5 nm away from the NT edge to mimic strong quenching at these very short distances, the simulations clearly reproduce the experimentally observed shifting of the emission positions in Figure 3. In all cases (Figure 4b−e), many dye
contrast, when dye molecules adsorb near gold NTs, a strong change in the emission map is observed.26 In the four experimental configurations where intensity enhancement was observed in Figure 2d−g (Cy5.5 with 640 nm excitation, Cy5 with 640 nm excitation, Cy5 with 561 nm excitation, and Cy3 with 532 nm excitation; Figure 3b−e, respectively), the NT acts as a nanoantenna to change the emission pattern: the emission from nearby molecules is modified by coupling to the plasmonic particle, resulting in a potential mislocalization between actual and apparent position in the far field. Indeed, though molecules that actually are on top of the NT will be quenched and not be detected in the fluorescence microscope, in all four cases, the maximum emission enhancement is observed from molecules that appear from their fits to be on top of the NT, clearly demonstrating the effect of coupling on the far-field radiation pattern. In fact, even when very little enhancement is observed (Figure 3d,e), most of the fluorescence fits are located on top of the NT (Figure S4), and fewer molecules appear from their fits to be at the periphery of the NT, indicating that the emission from molecules physically located around the NT is overall shifted toward the NT center. This shifting of the emission toward the NT center happens even when very little enhancement is observed (Figure 3d,e) and for molecules located up to ∼100 nm away from the NT, illustrating the robustness of this effect. Additionally, the resulting radiation pattern clearly reproduces the triangular shape of the NTs, giving us structural information on the substrate that is masked by the diffraction limit in standard optical microscopy. On the other hand, in the case of Cy3 with 515 nm excitation, where no emission enhancement is observed, the maps show that fewer molecules appear to emit 1737
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top of a glass coverslip sputter-coated with 100 nm of indium tin oxide. After developing the resist, a 5 nm Ti wetting layer and a 50 nm Au layer were evaporated onto the structure, and the remaining PMMA was lifted off. The resulting NTs were imaged using a high-resolution scanning electron microscope (Hitachi SU8000 In-Line), revealing triangles with rounded tips. Dark-Field Microscopy. A broadband halogen lamp was used to excite multiple NTs within an array through an oilimmersion dark-field condenser. The resulting scattered light was collected through a 100× oil-immersion dark-field objective with 0.6 NA and spectrally resolved using a spectrograph (Princeton Instruments, Acton SP-300i) coupled to an Andor iXon EMCCD camera. The dark-field signal measured on bare ITO was subtracted from this scattering signal, and this background-subtracted spectrum was then normalized by a spectrum of the halogen lamp source detected with the objective in bright-field mode (1.3 NA). PAINT Microscopy.30 Single-molecule wide-field epifluorescence microscopy was performed in an Olympus IX-71 inverted microscope. Three cyanine dye solutions were used successively in our experiments: Cy3, Cy5, and Cy5.5 prepared in concentrations of ∼2−200 nM in water. To avoid crosscontamination from one experiment to the next, the sample was rinsed with acetone, then soaked in acetone overnight to remove residual dye molecules. Additionally, any molecules remaining on the surface were photobleached by exposing the sample to a higher power laser beam (∼400 W/mm2, i.e., 10 times our typical (5−40 W/mm2) single-molecule fluorescence experiments) before the start of each experiment. In order to check that the sample was not damaged by these higher laser powers, we measured the dark-field scattering spectrum of each triangle array before and after fluorescence experiments and confirmed that the triangle plasmon resonances had not changed. The sample was illuminated through a 100×, 1.4 NA oil-immersion microscope objective by a 30 μm2 beam spot of circularly polarized CW light. The single-molecule fluorescence was collected through the same objective, and the image was projected via a 3× beam expander onto an Andor iXon EMCCD camera. Four different laser wavelengths were used, 515 nm (Spectra-Physics Excelsior 515-50-CDRH), 532 nm (CrystaLaser CL-532-025-O), 561 nm (Coherent Sapphire 561-50), and 640 nm (Coherent CUBE 640-40C), with excitation powers far from saturation (5−40 W/mm2) so that the intensity enhancement factors did not depend on this parameter. An appropriate long-pass filter (Semrock BLP01515R, BLP01-532R, BLP01-561R, and BLP01-640R, respectively) and dichroic mirror (Semrock Di01-R515, Di01-R532, Di01-R561, and Di01-R640, respectively) in the collection pathway rejected scattered laser light. During the course of an experiment, data were collected on all NT sizes with a constant excitation power. The ITO reference data were obtained by moving the beam spot between two NT arrays, keeping excitation and detection parameters the same as those used on the NTs. A reference movie was collected between each NT experiment to make sure that factors such as evaporation of the dye solution or sample heating were not affecting the enhancement factors. The enhancement factors were obtained by calculating the ratio of the average fluorescence intensity in the coupled dye−NT experiments (Figure 2b, red) to the average fluorescence intensity of the dye molecules on a reference ITO sample (Figure 2b, black).
molecules at the NT periphery appear to emit from on top of the NT. For the case of Cy5 coupled to 130 nm NTs (Figure 4c), the simulated intensity enhancement is plotted in Figure 4f against the magnitude of the mislocalization error (the difference between the actual dipole position and the fitted position). Consistent with our experimental results, a strong enhancement correlates with a large shift in apparent position; however there is still significant shifting even in the absence of enhancement. In the simulations, the highest emission intensity appears to come at the NT center position, where the highest density of fits is also recorded both experimentally and computationally (Figures S4 and S6, respectively). The simulations reveal that this mislocalization effect comes from the combined effect of the emission of dyes close to the antenna shifting toward the NT and the emission of dyes far from the antenna predominantly shifting away (Figure S7), resulting in a low density of localizations in the intermediate region. This combination of shiftingboth toward and away from a plasmonic antennawas previously reported in a system where the shifting was attributed to an interference effect between the source and its induced image dipole.34 Because the mislocalization in our system depends strongly on the local surface plasmon mode, the image dipole interpretation does not fully describe the results here. Overall, these simulations of emission enhancement agree well with our experimental results, reproducing the important features observed in the super-resolution maps: an increased density of localizations near the NT center, a decreased density of localizations near the NT edge, and maximal fluorescence enhancement for molecules localized near the NT center. In this Letter, we have examined the coupling between single dye molecules and plasmonic gold nanoparticles by varying the laser excitation wavelengths, dye emission and absorbance spectra, and local surface plasmon resonance frequency. The relative contributions of excitation and emission enhancement were measured, and we identified situations where excitation enhancement is critical (Figure 2d,e), situations where only emission enhancement plays a role (Figure 2f,g), and situations in which no enhancement is achieved (Figure 2h). However, although we verify that the excitation wavelength is a critical component of fluorescence enhancement, our super-resolution imaging and computational results show that the coupling and shifting of the dipole emission position toward the NT can be reproduced mainly by considering only the emission. Indeed, although the maximum fluorescence enhancement is observed when both the excitation laser wavelength and the dye emission spectrum overlap with the LSPR spectrum (Figure 2), superresolution imaging reveals similar strongly shifted emission patterns that are independent of the amount of enhancement for all dyes in all the excitation conditions (Figure 3). The only exception is for the 515 nm laser excitation (Figure 3e), in which case the excitation wavelength is well below the LSPR. In addition to separating the respective effects of enhanced emission and enhanced excitation on the distance-dependent coupling of single molecules, we show here that large coupling occurs even in the absence of strong enhancement, illustrating the power of super-resolution techniques to investigate light− matter interactions at the nanoscale.
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METHODS Gold Nanotriangle Substrates. Arrays of NTs were patterned by electron beam lithography (JEOL JBX-6300FS) in a 100 nm layer of poly(methyl methacrylate) (PMMA) spun on 1738
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angles (from 30° to 90°, linearly spaced) corresponding to the minimum symmetry unit of the triangle (blue dots in Figure 4a). Because the closest dyes are strongly quenched in our experimental configuration, the closest dye position simulated was 5 nm. At each dipole position considered, simulations were run for each of two orthogonal dipole orientations. For each dipole orientation, a reference simulation was run without the NT and under the same conditions. The fields were recorded in a plane in the glass substrate below the dipole source, mimicking the effect of our objective lens. Modeling the Apparent Emission Position. The simulated near-field field distribution was propagated to the far field using a standard far-field projection. Only the components radiating at angles within the 1.4 numerical aperture of our objective lens were kept. The resulting distribution was focused to a plane, and at each emission wavelength considered (570, 670, and 700 nm), this diffractionlimited image was fit to a 2D Gaussian function to determine the apparent position and intensity. The fluorescence intensity was calculated from the integral of the Gaussian function and normalized by the corresponding reference simulation. To predict the experimental fit distributions that come from random dye adsorption positions, Figures 4b−e, S5, and S6 were generated by randomly sampling interpolated results. The results of simulations of dipoles at the points indicated in Figure 4a were first interpolated onto a high-density square grid of points within the area spanning the simulated dipole locations. That high-density grid was then randomly sampled to produce the fit positions shown. The highest intensity points are shown on top for clarity.
The images were recorded at 25 frames/s to acquire emission only from dye molecules adsorbed on the sample surface. The adsorption rate is proportional to the solution dye concentration, which was limited to guarantee that only one molecule is adsorbed at a time per diffraction-limited area. Each isolated single molecule was located in a background-subtracted image and fit to a 2D Gaussian function with the MATLAB function nlinfit. Super-resolution maps were plotted with a dot at each apparent emission position; in each experiment, the data from molecules near 100−130 NTs within the array were collapsed onto a relative coordinate system for improved statistics (Figure 3). Dye Characterization. Dye absorbance spectra were obtained on concentrated cyanine dye solutions in water, with a lamp UV−vis spectrometer (Thermo Evolution UV− vis). The fluorescence emission spectra were obtained through the microscope with a Princeton Instruments spectrometer (Acton SP2300i) coupled to an Andor iXon EMCCD camera on a drop of concentrated dye solution in water, placed on an ITO-coated coverslip. All fluorescence experiments were performed with circularly polarized laser excitation and excitation powers between 5 and 40 W/mm2. FDTD Simulations. Electromagnetic simulations were performed using the Lumerical FDTD Solutions software package. NTs were modeled as 50 nm thick Au triangles with rounded corners on top of a 5 nm thick Ti congruent triangle. All simulations were performed with the NT immersed in water and placed on a substrate of 100 nm thick ITO on top of a glass slab. The NTs were placed at the center of a 1 μm3 simulation volume; near the NT a fine-mesh grid with 3.375 nm3 cell volume was used. The true NT geometry parameters (Figure S1, Table 1) were determined by varying the simulated size parameters (corner radius and equilateral side length) and matching the simulated scattering spectra to the experimentally measured dark-field scattering spectra. Water was modeled with a constant refractive index of n = 1.333, k = 0, and glass was set to n = 1.5, k = 0. The frequency-dependent complex permittivities of all other materials were obtained by an analytical fit to experimental data: ref 35 for Au, ref 36 for Ti, and ellipsometry data for ITO.26 For consistency with dark-field spectroscopy experiments, the simulated scattering spectrum of each NT (blue lines in Figure 1a−c) was calculated by exciting the NT from above the water at normal incidence with a broadband plane wave. For consistency with fluorescence microscopy experiments, the electric field enhancement was simulated by exciting the NT from below the glass coverslip with a broadband plane wave. In both cases, the results from two orthogonal excitation polarizations were averaged. The electric field intensity, |E|2, was simulated around the NT and then normalized by the corresponding value in a reference simulation with no NT. In the analysis, we set |E|2 = 0 for points on top of the NT, as any dyes in physical contact with the NT are quenched and not detected experimentally.12 The average field enhancement value over a 500 × 500 nm area containing the NT was then calculated for each NT and each laser excitation wavelength (open triangles in Figure 2d−h). The dipole coupling results in Figure 4 were obtained using a broadband point dipole source (constant current) to represent a dye molecule. To reproduce the coupled emission as a function of the dipole position, near each NT we simulated dipoles at seven different distances from the NT edge (from 5 to 100 nm, logarithmically spaced) and for eight different
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsphotonics.6b00344. Additional details about the NT properties, optical measurements, and finite difference time-domain calculations (PDF)
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. Present Address †
Department of Physics, Applied Physics and Astronomy, Rensselaer Polytechnic Institute, 110 8th Street, Troy, New York 12180, United States. Author Contributions
E.W. carried out the experimental measurements and analyzed the data. B.I. performed electromagnetic simulations. E.W., B.I., and J.B. discussed all the results and developed the distancedependent coupled emission model. E.W. wrote the paper, which was edited by all authors. All authors read and approved the final manuscript. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work was funded in part through a National Science Foundation CAREER award (grant CHE-1252322) to J.S.B. and by the Materials Research Science and Engineering Center 1739
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ACS Photonics
Letter
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(MRSEC) program of the NSF, grant DMR-1120923. B.I. was supported by the National Science Foundation Graduate Research Fellowship Program (grant DGE-1256260). The samples were prepared at the Lurie Nanofabrication Facility, a member of the National Nanotechnology Infrastructure Network, which is supported in part by the National Science Foundation. Thanks to Vishva Ray for assistance with the ebeam lithography.
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DOI: 10.1021/acsphotonics.6b00344 ACS Photonics 2016, 3, 1733−1740
