Effects of Tuning Fluorophore Density, Identity, and Spacing on

Nov 17, 2015 - A triplet-state mediated super-resolution imaging technique is used to optically investigate the locations of single fluorescently labe...
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Effects of Tuning Fluorophore Density, Identity, and Spacing on Reconstructed Images in Super-Resolution Imaging of FluorophoreLabeled Gold Nanorods Karole L. Blythe, Eric J. Titus, and Katherine A. Willets* Department of Chemistry, The University of Texas at Austin, 102 East 24th Street, Austin, Texas 78712, United States Department of Chemistry, Temple University, 1901 North 13th Street, Philadelphia, Pennsylvania 19122, United States S Supporting Information *

ABSTRACT: A triplet-state mediated super-resolution imaging technique is used to optically investigate the locations of single fluorescently labeled double-stranded DNA (dsDNA) ligands bound to gold nanorods (AuNRs). Previous work on this system has shown significant apparent heterogeneity in dsDNA ligand binding across the AuNR surface, but the reconstructed images have yielded dimensions that are smaller than the underlying AuNR substrate. Here, we tune the fluorophore density and identity to determine if the properties of the fluorophore impact the reconstructed images. While lowering the fluorophore density on the AuNR surface reduces the probability of simultaneous emission events and fluorophore self-quenching, we do not observe an improvement in the reconstructed image size and ultimately suffer from under-sampling of the surface. As alternative strategies, the identity of the fluorescent label, and thus the photophysics of the system, was also changed and again, the size of the reconstructed images remained smaller than expected in nearly all cases. Lastly, we increased the dsDNA linker length to decrease any interactions between the fluorophore and the gold surface and found that while the increased fluorophore-nanoparticle spacing impacted the photophysics of the fluorophore, the reconstructed image sizes remained consistently smaller than expected. Thus, we conclude that the photophysics of the fluorescent tag is not the primary origin of the incorrect size of the super-resolution images. However, in all cases, we continue to observe significant heterogeneity in the reconstructed images, further supporting our hypothesis that ligand binding from solution is nonuniform across the AuNR surfaces.

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INTRODUCTION Gold nanorods (AuNRs) have become a widely used substrate in biosensing and nanomedicine because they can be functionalized with different ligands that allow the nanoparticles to target specific analytes or regions of interest.1−7 AuNRs are often used because of their local heating properties,6,8 relative chemical inertness,9 and easily tunable localized surface plasmon resonance (LSPR).6,8,10−12 Functionalized AuNRs have been previously used in applications such as bioimaging,1−5 drug delivery,4,13,14 and photothermal therapy2,5−7 after the AuNR is functionalized with a targeting ligand to achieve the desired site-specific imaging or treatment. Ligands can also be used to reduce in vivo toxicity1,10,15 and improve the stability of AuNRs.1,2 The ligands attached to the surface of the AuNRs play a vital role in increasing the versatility of how AuNRs are used in different applications. Most functionalized AuNR conjugates are prepared by mixing thiolated ligands and AuNRs in solution and allowing the ligands to spontaneously assemble on the AuNR surface by exploiting the formation of gold−thiol bonds.16−25 Ideally, uniform surface coverage of the ligands will occur on each © 2015 American Chemical Society

AuNR in the solution, preserving the solution stoichiometry in the case of multiple ligand types. However, evidence of heterogeneous ligand binding has been previously reported in the form of preferential binding of certain ligands at the ends of AuNRs, leading to end-to-end assembly of the AuNRs into chains.21−26 Bulk fluorescence techniques have also been used to show that 1:1 solutions of DNA labeled with two different fluorophores do not preserve the solution stoichiometry when the ligands bind to the gold nanoparticle surfaces. 20 Unfortunately, these studies only show the average behavior of the population and cannot probe heterogeneity at the single nanoparticle level. Therefore, it is necessary to develop a technique to study ligand binding heterogeneity on single AuNRs at the single particle, single molecule level. In previous work from our group, we have used superresolution fluorescence imaging to map the apparent location of fluorescently labeled double-stranded DNA (dsDNA) on the Received: August 27, 2015 Revised: November 13, 2015 Published: November 17, 2015 28099

DOI: 10.1021/acs.jpcc.5b08364 J. Phys. Chem. C 2015, 119, 28099−28110

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The Journal of Physical Chemistry C surface of AuNRs.18,19 In these experiments we used thiolated dsDNA as the ligand, while a reporter molecule, carboxytetramethyl rhodamine (TAMRA), tethered to the dsDNA, provides a fluorescent signal that is used to localize the position of each dsDNA. We use a triplet-state shelving technique to photoswitch the fluorescent tags between an emissive “on” state and a nonemissive “off” state, such that only a single fluorophore is emissive at a time.27,28 The emission from a fluorophore in the “on” state is fit to a two-dimensional (2D) Gaussian, eq 1, where I(x,y) is the spatially dependent intensity, z0 is the background intensity, I0 is the peak intensity, sx and sy are the widths of the Gaussian in x and y, and x0 and y0 represent the center position. We approximate the location of the emitter as (x0,y0), which indirectly provides the position of where a dsDNA ligand is bound to the surface of the AuNR. ⎧ ⎡ x−x ⎨− 1 ⎢ s 0 2 e⎩ ⎣ x ⎪

I (x , y ) = z 0 + I 0

⎪

(

) + ⎛⎝ 2

⎜

y − y0 ⎞2 ⎤⎫ sy ⎟⎠ ⎥⎦⎬ ⎭

the underlying AuNRs, Figures S1−S6).18,19 As such, we would expect any fluorophore/plasmon coupling caused by spectral overlap to be much weaker for our system than for the system studied by Biteen and co-workers. In our group, we recently attempted to address the size mismatch issue by using a more rigorous model to fit our data, based on describing the inherent luminescence from the AuNR as the sum of three mutually orthogonal emitting dipoles (known as the 3-dipole model).19,33,34 While the 3-dipole model provided several benefits over a two-dimensional (2D) Gaussian, such as correctly localizing the center position of the AuNR, more high-quality fits of the calculated fluorophore positions, and useful output parameters about the underlying AuNR 3-dimensional orientation,19 the size mismatch issue was not resolved. Thus, while we continue to use this more rigorous model for all subsequent fits to our data, we cannot ascribe a poor choice of model or poor fitting as the origin of the size mismatch issue. In this manuscript, we discuss several experimental attempts to explore the size mismatch issue by modifying the photophysics of our samples. The first two strategies involve lowering the probability of multiple fluorophores emitting simultaneously. If multiple emitters are in the “on” state at the same time, we fit a superposition of that emission, which yields an intensity-weighted position that does not reflect the true position of each individual emitter. For fluorophores spaced on opposite sides of the AuNR, this superposition effect will move the calculated fluorescence position toward the center of the nanorod, leading to a smaller than expected size in the reconstructed nanorod image. For the triplet-state-mediated photoswitching strategy that we use in our experiments, we want all but one of the fluorophores shelved in the nonemissive triplet state at a given time, so that only a single emission event occurs within a given diffraction-limited spot. Experimentally, we have tried to control this by using high enough laser intensity to promote intersystem crossing (without melting the AuNR) and by working under a nitrogen environment to eliminate triplet-state scavenging oxygen so the molecules will reside longer in the triplet state. In other triplet-state-mediated photoswitching work, thiols are added to an oxygen scavenging buffer to help create even longer-lived dark states, by promoting formation of metastable dark species.35−37 However, in our system, introducing thiols interferes with the binding of the thiolated dsDNA to the gold surface, as evidenced by lack of fluorescence activity (vide infra). Unfortunately, this leaves us with a limited number of experimental variables to adjust to modify the triplet state of the TAMRA fluorophore. Therefore, we tested several other ways to lower the probability of emitters fluorescing simultaneously, both by changing the number of fluorophores tethered to the AuNR and by changing the identity of the fluorophore from TAMRA to one with a longer expected triplet state lifetime, Atto 532 (fluorescence maximum is ∼550 nm, Figure S7). As an alternative strategy to tuning the photophysics of the TAMRA label, we also increased the length of the dsDNA linker by 20 base pairs to test if the proximity to the AuNR surface was impacting fluorophore photophysics. Not only does this strategy allow us to determine whether we are sufficiently close to the nanoparticle surface to perturb the behavior of the fluorophore but also it is allows us to explore the possibility of plasmon coupling effects, which are expected to be distant-dependent. In all cases, we found that the reconstructed image sizes were smaller than the expected dimensions of the AuNR, indicating that dye photophysics is

⎪

⎪

(1)

This procedure is performed repeatedly to attempt to probe all the fluorescent labels bound to the AuNR surface as they switch between the nonemissive and emissive states. We use the calculated fluorophore positions to build reconstructed images of individual labeled AuNR substrates, and we have observed nonuniform distributions of calculated fluorescent positions with respect to the AuNR surface, suggesting binding heterogeneity of the dsDNA on the AuNR surface.18,19 However, while the reconstructed images of each AuNR show excellent agreement with the shape and orientation of the underlying AuNRs, we have found that the size of the optically reconstructed AuNRs are smaller than expected based on correlated AFM or bulk electron microscopy measurements.18,19 Other groups have observed similar size mismatch issues and skewing of calculated fluorescence positions that are located near a metallic surface when using super-resolution imaging. For example, research conducted by Uji-i and coworkers has indicated that the point spread function (PSF) of the fluorophore can be distorted because of its proximity to a metal nanowire surface, and these distortions will lead to inaccuracies when fitting a PSF image.29,30 In their work, the distortions were nanowire width dependent, leading to reconstructed images that were typically larger than expected based on the true nanowire dimensions.29 However, we do not visually observe the large distortions in the raw PSF data collected in our experiments on either nanowires31 and nanorods,18,19 suggesting that the smaller width dimensions of our nanowires and nanorods may help minimize the effect of the PSF distortions.18,19,31 Moreover, the rigorous fitting procedure we use (described in detail below and elsewhere)18,19 prevents significantly distorted PSFs from being included in our fits. Biteen and co-workers have observed the superlocalized position of Cy5.5 fluorescent molecules being skewed toward the center of the nearby gold nanoislands for distances up to 90 nm.32 The skewed position is thought to be caused by strong coupling between the fluorophore and the gold nanoisland due to the spectral overlap of the fluorophore emission and the localized surface plasmon resonance (LSPR) of the gold nanoislands. On the contrary, our experiments have been designed to minimize the spectral overlap between the reporter molecule emission spectrum (TAMRA emission maximum is ∼580 nm) and the LSPR maximum of the AuNRs (ranging from 630 to 700 nm, based on the dark field scatter spectra of 28100

DOI: 10.1021/acs.jpcc.5b08364 J. Phys. Chem. C 2015, 119, 28099−28110

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μL of 0.01% sodium dodecyl sulfate (SDS). After the final resuspension, the functionalized AuNR solution was stored in the refrigerator. In order to lower the density of fluorophores attached to AuNRs, we changed the concentrations of labeled and unlabeled dsDNA available in solution for binding to the AuNRs. Our initial concentrations of dsDNA used for experimentation was 7.2 nM of labeled dsDNA and 7.2 μM of unlabeled dsDNA for a total dsDNA concentration of 7.2 μM. When using these concentrations, the ratio of labeled dsDNA to unlabeled dsDNA was 1:1000. For comparison, we lowered the concentration of labeled dsDNA by an order of magnitude to achieve a concentration of 0.72 nM (resulting in a ratio of 1:10000 labeled dsDNA to unlabeled dsDNA). It should be noted that the concentration comparison was only done with TAMRA-labeled dsDNA. The experiments involving changing the fluorophore identity used 7.2 nM of Atto 532 dsDNA and 7.2 μM of unlabeled dsDNA (1:1000 labeled:unlabeled). Also, the 48 bp study was only done using TAMRA as the fluorophore at the 1:1000 labeled:unlabeled ratio. Coverslip Preparation. Two coverslip (25 × 25 mm no. 1 thickness glass) preparation protocols were used in these experiments. For the first preparation, the slides were cleaned with argon plasma and placed in a 0.5% (3-aminopropyl) triethoxysilane (APTES) in ethanol. The APTES helps the AuNRs stick better to the glass surface, which is important for subsequent atomic force microscopy (AFM) imaging. The slide was rinsed with ethanol and nanopure water and dried under nitrogen. Five microliters of diluted functionalized AuNRs (20:100 dilution in 0.01% SDS) was deposited on the slide and allowed to sit for 1 min before the slide was rinsed with nanopure water and dried under nitrogen. Lastly, 5 μL of diluted (1:50 dilution in nanopure water) sky blue fluorescent polystyrene beads (Spherotech) were deposited and allowed to sit for 45 s before the slide was rinsed and dried. For the second coverslip preparation, coverslips were cleaned with argon plasma and then the slides were rinsed with ethanol and nanopure water. After drying the coverslip under nitrogen, 5 μL of diluted (20:100 dilution in 0.01% SDS) functionalized AuNRs were deposited on the slide and allowed to sit for 5 min. Excess AuNRs were rinsed off with nanopure water, and the coverslip was dried under nitrogen. Finally, 5 μL of diluted (1:50 dilution in nanopure water) was deposited, and the excess was rinsed off after 5 min. The coverslip was dried under nitrogen. Optical Microscopy. Optical experiments were conducted using an Olympus IX-71 inverted microscope with an Olympus oil-immersion 100× objective with a variable numerical aperture (NA) between 0.6 and 1.3 (achieved by the objective’s internal iris). 532 nm laser excitation was passed through a quarter wave plate (to achieve quasi-circularly polarized light at the sample plane) and then a lens at the back of the microscope (to achieve wide-field illumination at the sample plane). A wide-field epi-illumination geometry was used to image the functionalized AuNRs. After passing through the lens, the laser excitation was reflected off a 532 nm dichroic beam splitter (Semrock), passed through the objective (NA set to 1.3), and was incident upon the sample. The laser intensity at the sample was ∼29 kW/cm2. Emission from a fluorescent label and the AuNR were collected back through the objective, passed through a long pass filter (Semrock), and imaged on an EMCCD (Princeton Instruments, PhotonMax or ProEM). The

not the primary origin of the size mismatch. However, we continue to observe significant particle-to-particle heterogeneity in the apparent dsDNA binding, suggesting that despite the size mismatch issue, our technique is sensitive to local ligand heterogeneity on nanoscale surfaces.

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EXPERIMENTAL METHODS Gold Nanorods. Cetyltrimethylammonium bromide (CTAB)-capped gold nanorods were synthesized using a seed-mediated technique.18,19,38,39 The CTAB-capped AuNRs were then coated with a thin layer of gold to cover any silver at the surface in order to create an ideal surface for thiol-gold binding chemistry.16 Briefly, a sufficient amount of chloroauric acid (in solution) was reduced by ascorbic acid (in solution). CTAB-capped AuNRs were centrifuged (10000 rpm for 20 min) once and resuspended in 18.2 MΩ cm resistivity nanopure water to remove excess CTAB. This solution was then centrifuged a second time, and the pelleted AuNRs were resuspended in sufficient 0.1 M CTAB solution to reach a AuNR concentration of 900 pM. Next, the reduced gold solution was mixed with AuNRs, and the solution was allowed to sit at room temperature to allow the reduced gold to bind to the AuNRs.16,18,19 Functionalizing Gold Nanorods with dsDNA. The functionalization protocol, based on a protocol published by Mirkin and co-workers,16 has been described in detail previously by our research group and will be briefly summarized here.18,19 The DNA used for functionalization was purchased as single-stranded DNA (ssDNA) from Integrated DNA technologies. Seven ssDNA were used to create the dsDNA used in the experiments discussed in this manuscript. For the 28 base pair (bp) dsDNA studies, the strands were TAMRA ssDNA (3′-[TAMRA]TTCTTAAATATTCGTCTTTTTTTTTTTT5′), Atto 532 ssDNA (3′-[Atto532]TTCTTAAATATTCGTCTTTTTTTTTTTT-5′), unlabeled ssDNA (3′TTCTTAAATATTCGTCTTTTTTTTTTTT-5′), and thiolated ssDNA (5′-AAGAATTTATAAGCAGAAAAAAAAAAAA[thiol]-3′). For the 48 bp dsDNA studies, the strands were 48 bp TAMRA ssDNA (3′-[TAMRA]TTCTTAAATATTCGTCTTTTCTTAAATATTCGTCTTTTTTTTTTTTTT5′), 48 bp unlabeled ssDNA (3′-TTCTTAAATATTCGTCTTTTCTTAAATATTCGTCTTTTTTTTTTTTTT-5′), and 48 bp thiolated ssDNA (5′-AAGAATTTATAAGCAGAAAAGAATTTATAAGCAGAAAAAAAAAAAAAA[thiol]-3′). The TAMRA ssDNA (or Atto 532 ssDNA) for the 28 bp and 48 bp dsDNA studies were hybridized with the thiolated ssDNA to create the labeled dsDNA. The thiol on one end of the dsDNA binds to the AuNR surface, while the fluorescent label is located away from the AuNR surface on the other end. The unlabeled dsDNA, for either dsDNA linker length, was created by hybridizing the unlabeled ssDNA and the thiolated ssDNA. Ideally, the unlabeled dsDNA functions as structural support for the labeled dsDNA, as well as provides spacing between the fluorescent labels to avoid self-quenching. Next, 100 μL of the overgrown AuNRs were centrifuged (10000 rpm for 20 min) twice. After the first centrifugation, they were resuspended in nanopure water, and after the second, they were resuspended in 50 μL of labeled dsDNA and 50 μL of unlabeled. Aliquots of sodium chloride were added to electrostatically screen the dsDNA, ideally resulting in wellpacked dsDNA on the AuNR surface. The mixed solution was allowed to sit overnight before undergoing three rounds of centrifugation (10000 rpm for 20 min) and resuspension in 100 28101

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position is extracted. A quality-of-fit threshold, R2 ≥ 0.8, is used to discard emission events that cannot be well-modeled. Each movie is processed separately and the average luminescence center position is used as a benchmark to combine the results of each movie into a single reconstructed image. The fluorescent polystyrene beads deposited on the slide serve as alignment markers.19,40 The emission from the beads (at least two per region) are fit with eq 1 so that their positions can be tracked for each frame of every movie. This position information is used to account for any mechanical drift that has occurred over the course of data collection.

data was taken using a 33 ms/frame integration time (with the PhotonMax) or 23 ms/frame integration time (with the ProEM). The discrepancy in integration times is due to the ability of the ProEM to achieve faster integration times than the PhotonMax. In both cases, each frame contained 356 × 356 pixels, and 3650 frames were collected per movie. Dark field microscopy was used to obtain scattering spectra for the AuNRs. The built-in overhead halogen lamp of the microscope was used as the light source, and an Olympus dark field condenser was used such that high-angle white light was incident upon the functionalized AuNRs. The NA of the objective was lowered to 0.6 in order to collect only low-angle scattered light. Scattered light from the underlying AuNR was collected using a spectrometer (Acton Research Corporation SpectraPro-150 or Princeton Instruments 2500i) and liquid nitrogen cooled camera (Princeton Instruments, Spec-10). Spectra were taken using 60 s integration times with or without three accumulations, depending on which spectrometer/camera system was used. All optical microscopy experiments were done under a constant nitrogen flow. Data Analysis. The data analysis process has been previously described in detail and will be summarized here.18,19 Either 3649 or 3650 frames of raw data were converted into TIFF stacks. The discrepancy in the frame number depends on which camera was being used for imaging; due to a timing error, the first frame of data taken with the PhotonMax had to be discarded (this was not an issue with the ProEM). Multiple movies were taken for each AuNR. The TIFF stacks were processed using a house-written MATLAB code. First, subsequent frames were subtracted to distinguish “on” (AuNR luminescence + fluorophore fluorescence) frames from “off” (AuNR luminescence only) frames. If subtraction of adjacent frames yielded intensity then the frame was marked “on”; otherwise it is marked as an “off” frame. Next, the intensity values of the “off” frames were placed into a histogram and only frames with intensities lower than one standard deviation above the mean were kept. This step rejects frames that are mistakenly assigned as “off” despite having a sizable contribution from the fluorophore. After making this initial cut, the intensities of the remaining “off” frames are placed into another histogram and fit with a Gumbel distribution. Only the frames with intensities lower than one standard deviation from the mean of the Gumbel distribution are kept. This analysis further rejects frames in which weakly emitting fluorophores are present and ensures that only frames in which AuNR luminescence is the contributing signal are assigned as “off” frames. Next, five “off” frames, chosen randomly over the entire movie, are modeled using the 3-dipole code.19,33,34 Only five frames were chosen due to the computational expense (∼18 min/frame) of using this rigorous modeling technique.34 After each frame is fit, an average AuNR contribution is calculated (). < AuNRfit> is corrected for any mechanical drift at each frame by using the Spherotech Sky Blue beads on the surface as alignment markers (see below).19 To assign “on” frames, we choose frames in which the emission intensity is five standard deviations above the average “off” frame intensity. Frames that cannot be marked definitively as “on” or “off” are left out of the data analysis. The driftcorrected < AuNRfit> is subtracted from each of the “on” frames, leaving only the fluorescence from the reporter molecule (either TAMRA or Atto 532). The remaining fluorescence is fit to a 2D Gaussian (eq 1) using a bounded least-squares fitting method, and the Gaussian center-of-mass

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RESULTS AND DISCUSSION Tuning the Density of Fluorophore-Labeled dsDNA. Previous Monte Carlo simulations from our group have shown that when the density of fluorescently labeled dsDNA is too high on the AuNR surface, the probability of multiple simultaneous emission events increases, leading to calculated emission positions that reflect an intensity-weighted superposition of the emitters that are skewed toward the geometric center of the AuNR.41 As a result, the simulations suggest that high numbers of fluorescent labels will lead to a smaller than expected size of the AuNR, consistent with our experimental results (Figure S8). To test this, we altered the concentration of fluorescently labeled-dsDNA available to bind to the AuNRs in solution. We started by optically investigating functionalized AuNRs prepared from the 1:1000 TAMRA-dsDNA:unlabeleddsDNA ratio solution. This ratio has been used in previously reported results by our research group and has yielded robust maps of calculated TAMRA emission positions.19 Unfortunately, the size of the reconstructed images were smaller than expected when taking into account the AuNR size and the dsDNA linker length (∼9.5 nm). Figure 1 shows the reconstructed images created using the 1:1000 ratio of TAMRA-dsDNA:unlabeled-dsDNA. The spatial frequency histogram maps, created by binning the calculated positions from TAMRA emission into 4.4 × 4.4 nm (1/10th of a pixel) bins and counting the number of points in each bin, are used to depict how the localization of TAMRA emission positions differ over the AuNR surfaces. The color scale bar next to the spatial frequency maps indicates how many positions are counted in each bin. The white “×” represents the luminescence center of the AuNR, as calculated from fitting the luminescence using the 3-dipole model.19,34 The spatial frequency maps in Figure 1 largely show rodlike reconstructed images that reveal the shape and orientation of the underlying AuNRs, as well as the spatial distribution of the TAMRA dye activity on the AuNR surface. We have previously shown excellent agreement between the orientation of the reconstructed images and correlated AFM images; thus, we are confident that these spatial intensity maps reflect structural features of the underlying AuNR.18,19 Moreover, the nanorod orientation that is extracted from the 3-dipole fit to the AuNR luminescence (orange ovals) matches the orientation of the reconstructed images, providing confirmation that the reconstructed images reflect the orientation of each underlying AuNR substrate.19,34 However, as with previous work, we find that reconstructed images are smaller than the expected dimensions of the AuNRs used in this study (AuNRs are 86 ± 7 nm long by 28 ± 3 nm wide; accounting for the ∼9.5 nm dsDNA spacer length should yield final dimensions of ∼105 × ∼47 nm, as shown by the orange ovals in Figure 1). 28102

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frequency maps, as well as the fit to the 3-dipole code which suggests a major axis in the luminescence emission.34 For the majority of spatial frequency histogram maps in Figure 1, the AuNR calculated luminescence position is centrally located with respect to the TAMRA positions, which is expected for TAMRA-dsDNA being distributed over the AuNR surface and verifies that the white × overlaps quite well with the geometric center of the AuNR.19 However, in Figure 1K, most of the localized TAMRA emission events are located below and to the right of the AuNR luminescence center position, providing evidence that TAMRA-dsDNA bound only to the lower half of this AuNR. If the nanoparticle were in fact spherical, the white × would lie in the center of the calculated TAMRA positions. Figure S9 summarizes several different DNA binding mechanisms that could lead to asymmetric spatial intensity maps. To probe whether any local fluorescent enhancement was present in these samples, we calculated spatial intensity maps, in which we plot the average intensity of the TAMRA emission events in a particular bin (Figure S10). The spatial intensity maps show that we are not observing intensity enhancement across the AuNR surfaces that would indicate surface-enhanced fluorescence and therefore fluorophore-AuNR coupling. This result is consistent with our expectations because we have purposefully minimized the overlap between the fluorescence emission and the scattering longitudinal plasmon mode of the AuNR (Figure S7). Next, we lowered the concentration of TAMRA-dsDNA by an order of magnitude, using a ratio of 1:10000 TAMRAdsDNA:unlabeled-dsDNA in solution that was mixed with the AuNRs. When using this dilution level, we expect to see very low numbers of binding events occurring on the AuNRs, meaning that there should be very few TAMRA emission events to fit. By lowering the number of TAMRA-dsDNA on the surface, we lower the probability of TAMRA molecules emitting simultaneously, allowing us to probe whether simultaneous emission events lead to smaller-than-expected reconstructed images. Figure 2 shows examples of reconstructed images created from imaging the functionalized AuNRs prepared with the lower concentration of TAMRAdsDNA. The spatial frequency histogram maps were created using 4.4 × 4.4 nm bin sizes for Figure 2, panels A−C and E− G, and 5.3 × 5.3 nm for Figure 2, panels D and H−K. Both bin sizes correspond to 1/10th of an imaging pixel and are only dependent on which EM-CCD camera was used for data collection. The spatial frequency maps in Figure 2, panels A−D, show rodlike reconstructed images. This concentration level should lead to fewer than one fluorescent label per rod based on calculations of the average DNA footprint on a nanorod surface.17 Thus, these results suggest cooperative binding, in which it is possible that once a TAMRA-dsDNA binds, it helps facilitate binding of the same type of dsDNA, leading to a nonstoichiometric number of TAMRA-dsDNA binding events on the AuNR relative to the unlabeled dsDNA. The remainder of the reconstructed images, Figure 2, panels E−K, show sparse spatial frequency maps which are not immediately obvious as rods. The small number of TAMRA emission events localized across the surface of the AuNRs may indicate few binding events, which is not surprising based on the low concentration of TAMRA-dsDNA that was used. However, if we use the average size of the AuNRs and the orientation calculated from the 3-dipole fit to the AuNR luminescence, we find that the

Figure 1. Reconstructed images created using the 1:1000 ratio of TAMRA-dsDNA:unlabeled-dsDNA. The spatial frequency maps in this figure suggest several different behaviors: (A−D) evenly distributed TAMRA-dsDNA binding across the AuNR surfaces, (E− F) preferential binding of TAMRA-dsDNA toward the ends of the AuNRs, and (G−K) asymmetric binding favoring one end over the other. The white “×” represents the average-calculated AuNR luminescence position for each example. All images share a common 50 nm scale bar. The bin size in the reconstructed images equals 4.4 × 4.4 nm. Orange ovals show the expected size of the functionalized AuNRs based on average AuNR dimensions and the dsDNA linker length, ∼105 × 47 nm. The orientation of the oval represents the angle of the long-axis of the underlying AuNR, as calculated from the 3-dipole fit to the AuNR luminescence.

Figure 1 also reveals the heterogeneity in where the TAMRA positions are localized over the different AuNR surfaces. We hypothesize this is caused by binding heterogeneity that occurs when dsDNA attaches to the AuNRs in solution during the assembly process and is in agreement with previous results.18,19 Several different types of behaviors are observed: for example, the spatial frequency histogram maps in Figure 1, panels A−D, show evenly distributed fluorescence positions in the shape of the underlying AuNRs, suggesting that uniform binding of the fluorescently labeled dsDNA occurred across the AuNR surface. Figure 1, panels E and F, show examples where the majority of calculated emission positions were localized at the ends of the AuNRs with fewer points localized at the center, suggesting preferential binding of the TAMRA-dsDNA to the AuNRs ends, consistent with other literature reports.21−26 The spatial frequency maps in Figure 1, panels G−K, show more TAMRA emission localization events on one end of the AuNR than the other. This is especially obvious in Figure 1K, in which the reconstructed image appears more consistent with a spherical nanoparticle rather than a rod. However, we are confident that this is a rod with incomplete binding across the AuNR surface. This conclusion is supported by the center position of the AuNR luminescence, marked with a white × in the spatial 28103

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Table 1. Number of “On” Events Fit Normalized by the Amount of Frames Acquired for Each of the Fluorophore Identity and Concentration Combinations identity of labeling fluorophore

base pairs (bp) in dsDNA linker

ratio of labeleddsDNA:unlabeleddsDNA

TAMRA TAMRA TAMRA Atto 532 TAMRA

28 28 28 28 48

1:10000 1:1000 1:1 1:1000 1:1000

number of “on” events fit per frame (average ± standard deviation) 0.0025 0.028 0.0097 0.022 0.011

± ± ± ± ±

0.0026 0.014 0.0151 0.012 0.007

bursts/frame for the 1:1000 TAMRA-dsDNA:unlabeleddsDNA concentration, and a drop to 0.0025 bursts/frame for the 1:10,000 TAMRA-dsDNA:unlabeled-dsDNA concentration. This order of magnitude drop in fluorescence activity shows that the super-resolution studies (on average) reflect the order of magnitude drop in fluorophore coverage, indicating that we are well away from the regime in which interdye fluorescence quenching would occur. We also optically investigated functionalized AuNRs prepared by mixing a 1:1 ratio of TAMRA-dsDNA:unlabeleddsDNA (3.6 μM each). In this high concentration regime, we expect that interdye fluorescence quenching can occur. Table 1 shows that the average number of fluorescent bursts per frame is 0.0097 in this regime, which is lower than the activity of the 1:1000 TAMRA-dsDNA:unlabeled-dsDNA sample and strongly indicates the presence of interdye fluorescent quenching in the 1:1 sample. The associated spatial frequency histograms are shown in Figure S14 and despite the internal quenching show significant heterogeneity across the different examples, with multiple AuNRs showing preferential binding at a single end. The spatial frequency maps also show that the reconstructed images are smaller than expected, consistent with the trends observed for the lower concentration samples. Our concentration studies ultimately show that the 1:1000 TAMRAdsDNA:unlabeled-DNA ratio appears to generate the best results, with no evidence of fluorescence quenching (given the order of magnitude activity drop as the concentration is changed by an order of magnitude) and nonsparse reconstructed images, yet even in this regime, the reconstructed images remain smaller than expected. Changing the Identity of the Fluorophore Label. The second experimental attempt for addressing the size mismatch issue was to change the fluorescent label to a molecule with a longer lived triplet “off” state. In this case, having the fluorophores in an off state longer lowers the probability of populating the singlet ground state. Therefore, we have a better chance of having only one fluorophore at a time in the “on” state. It has been reported in the literature that TAMRA, when adhered to a glass coverslip, has a triplet-state lifetime of only ∼3.1 μs.42 To achieve a longer off time, we switched the reporter molecule to Atto 532, which has a reported dark state time of ∼230 ms in poly(vinyl-alcohol) (unfortunately, we have been unable to find a direct comparison between the two dyes in the literature).27 The local environment [e.g., nitrogen, ambient air, poly(vinyl-alcohol), etc.] of the molecule can greatly affect its triplet-state lifetime; therefore, the lifetimes reported cannot be directly applied to our experiment. However, they can provide a clue to which molecules might be better-suited for the triplet-state-mediated imaging technique. The emission of Atto 532 is also blue-shifted relative to the

Figure 2. Reconstructed images created using the 1:10000 ratio of TAMRA-dsDNA:unlabeled dsDNA. The spatial frequency maps in A− D suggest evenly distributed dsDNA binding on AuNRs, creating rodlike shapes. The spatial frequency maps in E−K suggest that sparse TAMRA-dsDNA binding events occurred on the AuNR surfaces. The white × in the spatial intensity maps represents the average AuNR luminescence position for each example. All images share a common 50 nm scale bar. Bin sizes in the reconstructed images equal 4.4 × 4.4 nm for A−C and E−G and 5.3 × 5.3 nm for D and H−K. Orange ovals show the expected size of the functionalized AuNRs based on average AuNR dimensions and the dsDNA linker length, ∼105 × 47 nm. The orientation of the oval represents the angle of the long-axis of the underlying AuNR, as calculated from the 3-dipole fit to the AuNR luminescence.

points fall within the expected shape and orientation of the nanorod (orange ovals, Figure 2). The results of using this lower concentration of TAMRAdsDNA show that the reconstructed images are still smaller than expected. The corresponding spatial intensity maps for the spatial frequency maps in Figure 2 can be found in Figure S11 and again, we did not observe intensity increases across the surface of the AuNRs that would suggest surface-enhanced fluorescence and fluorophore-AuNR coupling. We also checked whether lowering the concentration of the TAMRA by an order of magnitude yielded a corresponding change in the number of fluorescent bursts associated with each nanorod. Given that more data was collected for some AuNRs than others, we normalized the number of fluorescent bursts that were successfully fit by the number of frames acquired (the average number of “on” events fit per frame for each data set is shown in Table 1, data for individual functionalized AuNRs are shown in the Tables S1−5 in the Supporting Information). For the 1:1000 ratio, we included data from previously reported results,19 using the same sample preparation, to calculate the average “on” events fit per frame. This was done for the purpose of representing all the data collected using these conditions. We found that, on average, we observed 0.028 28104

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efficiency of the AuNR and the quality of the reconstructed image. However, if we look for a trend across the entire data set (both Atto 532 and TAMRA), we find no relationship between reconstructed image size and AuNR plasmon resonance, dark field scattering intensity, or gold luminescence intensity. Moreover, the associated spatial intensity maps (Figure S12) show no spatial relationship with the fluorescence intensity, further supporting a lack of connection between the plasmonic properties of the nanostructure and the small reconstructed images. We also rule out an orientation artifact because there are similarly orientated rods that are smaller than expected in both length and width; thus, the size mismatch is not due to aberrations in the optics.19 Interestingly, we find that the activity of the Atto 532 at the 1:1000 labeling density is 0.022 bursts/frame, which is (at the 95% confidence level) statistically the same activity as the TAMRA at the same loading density (0.028 bursts/frame). Thus, our attempt to modify the photophysics of the system by trying a different fluorophore appears to yield nearly identical photophysical behavior. Using the Atto 532, we still observe evidence of heterogeneous binding of the labeled-dsDNA on the AuNR surface. The spatial frequency histogram maps in Figure 3, panels A−E, depict evenly distributed localization of Atto 532 emission across the surface of AuNRs, suggesting an even binding distribution of the labeled-dsDNA. The spatial histogram maps in Figure 3, panels F−H, suggests preferential binding toward the AuNR ends, and Figure 3, panels I−K, suggests instances where one end of the AuNR was preferred over the other end. The white × in the maps represents the average AuNR luminescence position for each example and are centrally located with respect to the Atto 532 emission data points, as expected and previously shown in the TAMRA 1:1000 data set. In addition, the corresponding spatial intensity maps are included in Figure S12 and S15 to show, again, that we did not observe local intensity increases that suggest surfaceenhanced fluorescence. Importantly, our results are quite similar between the two different fluorophores, indicating that the binding heterogeneity observed in the TAMRA data set is reproduced when using a different fluorescent label. Increasing the Distance between Fluorophore and AuNR. As an additional experiment, we increased the distance between the TAMRA molecule and the AuNR surface by increasing the length of the dsDNA linker to ∼16.3 nm (48 bp) from ∼9.5 (28 bp). For these experiments, we used the 1:1000 ratio of TAMRA dsDNA:unlabeled dsDNA because this mixture provided the best results for the shorter dsDNA (Figure 1). Figure 4 shows the reconstructed images created by mapping the location of emission from TAMRA molecules tethered to AuNRs by the longer dsDNA. Once again, the reconstructed images in Figure 4 are smaller than the expected dimensions of the AuNR, similar to the results in Figure 1 using the shorter dsDNA linker. However, we observe a significant decrease in the average number of “on” events fit per frame when comparing the longer dsDNA linker data set, 0.011 ± 0.007, with the shorter dsDNA linker data set, 0.028 ± 0.014 (Table 1). One possibility is that we have fewer-labeled dsDNA binding to the AuNR surface when using the longer dsDNA linker, similar to the reduction in activity observed when the proportion of labeled dsDNA in the sample was reduced from 1:1000 to 1:10000 (Table 1). On the basis of our 1:10000 dilution results (Figure 2), we would expect fewer labels on the surface to lead to more sparsity in the reconstructed images. However, the overall dimensions and coverage in the

TAMRA, moving us even further from the regime where overlap between the emission and longitudinal (scattering) plasmon mode of the AuNR occurs (Figure S7). The Atto 532 experiments were conducted using the 1:1000 ratio of Atto-dsDNA:unlabeled-dsDNA and the same dsDNA linker (∼9.5 nm long) as the TAMRA experiments. We chose this concentration regime because it yields nonsparse spatial frequency maps where no evidence of self-quenching occurs. Figure 3 shows a montage of reconstructed images created

Figure 3. Reconstructed images created using the 1:1000 ratio of Atto532-dsDNA:unlabeled-dsDNA. The spatial frequency maps in this figure suggest evenly distributed Atto 532-dsDNA binding across the AuNR surfaces (A−E), preferential binding of Atto 532-dsDNA toward the ends of the AuNRs (F−H), and more binding occurring on one end than the other (I−K). The white × represents the average AuNR luminescence position for each example. Images A and B share a common 50 nm scale bar, and images C−K share a common 50 nm scale bar. The bin size in the reconstructed images equals 4.4 × 4.4 nm. Orange ovals show the expected size of the functionalized AuNRs based on average AuNR dimensions and the dsDNA linker length, ∼ 105 × 47 nm. The orientation of the oval represents the angle of the long-axis of the underlying AuNR, as determined from the 3-dipole fit to the AuNR luminescence.

using the calculated positions from Atto 532 emission (more examples can be found in Figure S15). Encouragingly, one of the spatial frequency maps (Figure 3A) shows dimensions consistent with the size of the DNA-labeled AuNR in both the length and width dimensions, while a second example (Figure 3B) has strong agreement in the width dimension, although it somewhat underestimates the length of the AuNR. However, the remaining 17 examples (shown in both Figure 3 and Figure S15) have sizes well-below the expected dimensions of the AuNRs. Looking at the associated dark-field scattering spectra (Figure S4), we were initially encouraged by the weak scattering produced by the AuNR shown in Figure 3A, suggesting perhaps some relationship between the scattering 28105

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with some rods showing activity along the entire length, others showing increased activity on the ends, and some showing preferential activity on one side of the AuNR. To attempt to quantify this heterogeneity, we calculated an asymmetry factor (AF) for each of the reconstructed images. For this calculation, we first define the center of the AuNR using the calculated luminescence position from the 3-dipole fit to the AuNR luminescence background. Next, the reconstructed image of a functionalized AuNR (Figure S16A) is rotated around the AuNR luminescence center position such that the long axis of the AuNR reconstructed image is along the x axis (Figure S16B). The angle of rotation is based on the calculated in-plane angle determined by fitting the AuNR luminescence to the 3dipole model (same angle as the orange ovals in the spatial frequency maps from Figures 1−4). After the rotation, the number of points that lie positive and negative of the luminescence center are counted and applied to eq 2 to obtain the asymmetry factor: Figure 4. Reconstructed images created using the ∼16.3 nm long dsDNA and 1:1000 ratio of TAMRA-dsDNA:unlabeled-dsDNA. The spatial frequency maps in this figure suggest several different behaviors: (A and B) evenly distributed TAMRA-dsDNA binding across the AuNR surfaces, (C) preferential binding of TAMRAdsDNA toward the ends of the AuNRs, (D−G) asymmetric binding favoring one end over the other, and (H) sparse TAMRA-dsDNA binding events occurred on the AuNR surfaces. The white × represents the average AuNR luminescence center position for each example. All images share a common 50 nm scale bar. The bin size in the reconstructed images equals 4.4 × 4.4 nm. Orange ovals show the expected size of the functionalized AuNRs based on average AuNR dimensions and the dsDNA linker length, ∼119 × 61 nm. The orientation of the oval represents the angle of the long-axis of the underlying AuNR, as calculated from the 3-dipole fit to the AuNR luminescence.

AF =

#positive − #negative #positive + #negative

(2)

The asymmetry factor is calculated for both the length and width dimensions of the AuNR, using the points that lie positive and negative relative to the x and y axes, respectively (Figure S16B). The asymmetry factor for each functionalized AuNR discussed in this paper (as well as AuNRs studied in previous work)19 was calculated and can be found in Tables S1−S5. Figure 5 shows the histograms of the asymmetry factors, separated by fluorophore identity, concentration, and dsDNA linker length. For a AuNR uniformly covered with labeled-dsDNA, we would expect an asymmetry factor of 0; likewise, for an AuNR in which the ends are preferentially labeled with dsDNA, with minimal coverage in the center, we would also expect an asymmetry factor of 0. However, the distributions show a variety of asymmetry factors, with a sizable fraction with values of 0.5 or more, indicating that we are seeing a significant population where the fluorophore activity is biased toward one end of the AuNR. This trend is independent of fluorophore concentration and fluorophore identity, suggesting that the asymmetry is related to the binding of the DNA to the AuNR surface, as shown schematically in Figure S9. However, we do note that the examples using the longer dsDNA linker have asymmetry factors clustered around zero, with no examples showing high degrees of asymmetry. Given that all samples use AuNRs prepared under identical conditions, we cannot attribute this to a surface effect associated with the AuNR substrate and instead assign this as a ligand-based effect. We hypothesize that the longer linker allows more conformational flexibility on the surface, possibly allowing more homogeneous distributions of labeled versus unlabeled dsDNA on the AuNR. Importantly, this asymmetry is completely hidden in ensemble measurements or even measurements that study end-to-end linking of labeled AuNRs.21−26 Quantifying the under-Estimation of Size. Finally, we compiled all the examples for each of the functionalized AuNRs into separate cumulative distribution function (CDF) plots (Figure 6). These plots are constructed by counting the fraction of points that fall within a given distance of the AuNR luminescence center, both in the positive and negative direction relative to the length and width axes of the rotated AuNR (similar to the asymmetry factor calculation above). CDF plots,

reconstructed images in Figure 4 (1:1000 TAMRA:unlabeled, 48 bp DNA) agree quite well with the images in Figure 1 (1:1000 TAMRA:unlabeled, 28 bp DNA), suggesting that the reduction in activity is not due to significant loss of DNA binding to the surface. A second possibility is that the photophysics of the TAMRA is impacted by its distance to the gold surface. Previous work has shown that when a fluorophore is placed in proximity to a metallic surface, it is possible to achieve enhanced radiative and nonradiative rates.43−47 Geddes and co-workers have used this phenomenon to study enhanced triplet quantum yields and the metalenhanced phosphorescence of Rose Bengal in proximity to silver island films.43,44 Moreover, Kreiter and co-workers have observed the suppression of the fluorescent blinking from DiIC1(5) molecules when placed in proximity to gold thin films.45 Due to the metallic surface, the depopulation rate of the triplet state increases, leading to a reduction in blinking due to long excursions to the triplet state.45 In our case, when we use the shorter dsDNA linker, the metal surface could depopulate the triplet state more rapidly, leading to shorter triplet state lifetimes and thus more “on” events in comparison to the longer DNA linker. Unfortunately, this effect only manifests itself as a reduction in the number of on events with the longer dsDNA linker and does not change the size of the reconstructed dsDNA-AuNR image. Quantifying Asymmetry. In all of the examples presented in Figures 1−4, we have observed significant heterogeneity in where the TAMRA or Atto 532 activity is localized on AuNRs, 28106

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Figure 6. Cumulative distribution function plots depicting length and width data for all reconstructed images shown for each fluorophore identity, concentration, and dsDNA length increase (bp = base pairs) study. For each experimental condition (separated by rows): within a given cumulative distribution plot each color corresponds to a different AuNR, and between the length and width plots, each color corresponds to the same functionalized AuNR example. For the length CDF plots: × represents the values on the right side of the AuNR luminescence center and ◆ represents the values on the left side of center. For the width CDF plots × represents the values above the center and ◆ represents the values below. Distance increments of 4 nm were used to construct the plots.

Figure 5. Histograms of asymmetry factors, calculated for both length (left column) and width (right column), for each of the functionalized AuNRs discussed in this paper. Each row corresponds to a different fluorophore identity/concentration/dsDNA linker length data set.

although typically used for statistical modeling of data, have proven to be a convenient way to view and compare the size dimensions of the reconstructed image data for a whole data set on a single plot. The CDF plots are also a good way to visualize the heterogeneity of the calculated fluorophore positions localized across the surface of the AuNRs. In Figure 6, the CDF plots for the length and width dimensions of the reconstructed functionalized AuNR images for each experimental condition are shown. Unfortunately, fitting these curves to a model to extract meaningful, not arbitrary, length and width parameters has proven to be difficult because one model does not fit all of the

data due to the heterogeneity in our system. We have used the data from the Monte Carlo simulations (Figure S8) to see what the distributions in the CDF plots look like in ideal wellbehaved (simulated) cases, and the curves look linear over distances from the center of the AuNR to the expected halflength or half-width of the nanorod. In our experimental data, while some AuNRs show a somewhat linear CDF curve until 28107

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fluorophore proximity to the AuNR surace are not the source of the smaller than expected reconstructed image sizes. However, throughout all of the experiments, we found significant heterogeneity in fluorophore activity across the different reconstructed images, which is suggestive of heterogeneous binding of the dsDNA ligands to the surface of AuNRs. This result is hidden in traditional ensemble-averaged experiments and highlights the need for studying these systems at the single particle, single molecule level. We are still in the process of understanding the cause of the mismatch between the actual and reconstructed image size. Although we have not seen any evidence of fluorophore-AuNR coupling, such as locally enhanced fluorescence intensity, it is possible that being near a metallic surface is altering the lifetimes and photophysics of the fluorophore, as suggested by the decrease in activity when using the longer DNA linker. Therefore, it would be beneficial to perform fluorescence lifetime experiments to see if there is a difference between the lifetimes of free fluorophores in solution and the lifetime of fluorophore-dsDNA-labeled AuNRs in solution. We must also consider the possibility of image dipole formation between the fluorophore and AuNR, which can impact how light is radiated into the far field.48 Nevertheless, despite the consistent mismatch between the reconstructed and actual dsDNA-labeled AuNR sizes, evidence continues to mount in favor of ligandbinding heterogeneity on the surface of these important nanoscale materials.

reaching a final length and width value, indicative of more uniform coverage of TAMRA activity, others have a more sigmoidal shape, reflective of a AuNR with few emission positions located near the center of the rod. Thus, fitting the data to an arbitrary function to extract a final size of the AuNR has proven to yield nonquantitative results, especially when compared to the more straightforward, but less rigorous, “eyeball” analysis (i.e., estimating size by eye). The average size of the AuNRs used in these studies is 86 ± 7 nm long by 28 ± 3 nm wide (as determined by electron microscopy) and, therefore, we expect the functionalized AuNRs to be ∼105 × ∼47 nm after accounting for the ∼9.5 nm dsDNA spacer length. Ideally, this would be reflected in the length CDF plots by a plateau in the curve occurring ∼53 nm from the center. Unfortunately, for most of the experimental conditions (excluding the one Atto 532 example shown in Figure 3A), the CDF plots show that ∼100% of the data points are accounted for well below this value. In the width CDF plots, 100% of the points should be accounted for at a distance of ∼24 nm from the center on both sides of the AuNR luminescence center. While we see better overall agreement between the calculated and expected widths, we still find that the majority of the AuNRs have CDF plots that reach 100% of the calculated emitter positions well below the expected distance from the luminescence center. When using the longer dsDNA, ∼16.3 nm, we expect the functionalized AuNRs to be ∼119 × ∼61 nm. Therefore, 100% of the points should not be accounted for until ∼60 nm from the center for the length CDF plots and ∼31 nm for the width CDF plots. Unfortunately, there is not a noticeable difference between the plateau points in the CDF plots for the different experimental conditions, despite the change in the DNA spacer length. As a final point, we note that the CDF plots show significant particle-to-particle heterogeneity. While this has been obvious by comparing the individual spatial frequency maps in Figures 1−4, the CDF plots reveal this heterogeneity in a single plot, allowing us to visualize the extent of the heterogeneity in a single sample type. For example, the Atto 532 plots show much greater diversity in the CDF plots than the comparable 1:1000 TAMRA data, suggesting the possibility that this fluorophore can induce greater heterogeneity in local binding. To test this hypothesis, we plan to perform multiple labeling experiments, in which we compete identical dsDNA with different fluorophore labels to see if preferential binding continues to occur.

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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/acs.jpcc.5b08364. Dark field scattering spectra of AuNRs, TAMRA and Atto 532 emission spectra, Monte Carlo simulated reconstructed images, proposed mechanism for asymmetric spatial frequency histogram maps, spatial intensity maps, 1:1 TAMRA-dsDNA:unlabeled-dsDNA data set, additional 1:1000 Atto 532-dsDNA:unlabeled-dsDNA examples, and tabulated results showing heterogeneity and asymmetry factors for individual functionalized AuNRs (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected].

CONCLUSIONS In this manuscript, we described several experimental attempts to understand the size mismatch between the expected and observed dimensions of the super-resolution reconstructed images of fluorescently labeled dsDNA-functionalized AuNRs. The experiments focused on lowering the probability of having multiple molecules emitting simultaneously, as this can cause the size of the reconstructed images to shrink relative to the actual size of the dsDNA-labeled AuNR. We varied the TAMRA-dsDNA concentration and tested a different reporter molecule that has a reported longer lived “off” state. We also increased the distance between the fluorophore and the AuNR surface to investigate if photophysics were being altered due to proximity to a metal surface and if this could yield insight into the incorrect reconstructed image size. Unfortunately, the sizes of the reconstructed images were almost always smaller than expected, indicating that simultaneous emission events and

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The work presented in this chapter was supported by the National Science Foundation under Grant CBET-1402610, Welch Foundation Award No F-1699, and start-up funds from Temple University.

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REFERENCES

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DOI: 10.1021/acs.jpcc.5b08364 J. Phys. Chem. C 2015, 119, 28099−28110