Super-resolution imaging of single-molecule surface-enhanced Raman scattering (SM-SERS) reveals a spatial relationship between the SERS emission centroid and the corresponding intensity. Here, an isotope-edited bianalyte approach is used to confirm that shifts in the SERS emission centroid are directly linked to the changing position of the molecule on the nanoparticle surface. By working above the single-molecule limit and exploiting SERS intensity fluctuations, the SERS centroid positions of individual molecules are found to be spatially distinct.

Triplet-state-mediated super-resolution imaging was used to map the positions of fluorescently labeled double-stranded DNA bound to the surface of gold nanorods. In order to isolate individual fluorophores bound to the nanorod surface, imaging conditions were optimized such that the majority of the fluorophores were forced into a triplet dark state, and fluorescence from approximately one molecule at a time was detected. The fluorescence from the emitting single molecule was then fit to a two-dimensional (2D) Gaussian to localize its position relative to the nanorod substrate. The reconstructed super-resolution images showed excellent agreement with the shape and orientation of the nanorods, although, based on correlated atomic force microscopy, they consistently under-estimated nanorod size. The apparent DNA ligand binding on the gold nanorod surface showed significant heterogeneity, with examples of preferential binding to nanorod ends, uniform binding across the nanorod surface, and site-specific binding to a single end of the nanorod. This heterogeneity would be hidden in a typical ensemble or diffraction-limited measurement, highlighting the need for single nanoparticle super-resolution imaging studies.

We present a study comparing the accuracy of superlocalization imaging of plasmon-mediated emission from gold nanorods (AuNRs) using both Gaussian and dipole emission point-spread function (PSF) models. By fitting the emission PSF of single AuNR luminescence, we have shown that a 3-axis dipole PSF gives improved localization accuracy over the Gaussian PSF, especially for nonplanar AuNRs, while also allowing the AuNR three-dimensional orientation and emission wavelength to be determined. On the other hand, when a single-axis dipole PSF model is applied to the AuNR emission, the fit estimates converge to values that are inconsistent with their experimentally measured values, affecting both the localization accuracy and precision of the fitted centroid position. These results indicate that when applying superlocalization techniques to plasmonic nanostructures, care must be taken to understand the nature of the emission before a correct dipole PSF can be applied.

We use a triplet-state-mediated super-resolution fluorescence imaging technique to localize the position of individual fluorescently labeled double-stranded DNA (dsDNA) bound to the surface of gold nanorods. Within each diffraction-limited spot, we must account for two different emission sources: the stochastic fluorescence from the fluorescent labels and the steady background luminescence of the gold nanorod. To isolate the contribution from the fluorescent label, we subtract the average gold nanorod luminescence contribution, modeled with either a two-dimensional Gaussian or a dipolar emission model. The fluorescence from the labeled dsDNA is then fit with a two-dimensional Gaussian to reconstruct the positions of each individual emitter on the nanorod surface. The resulting reconstructed images, using either luminescence model, agree with the shape and orientation of the underlying nanorod, and show similar apparent dsDNA binding heterogeneity across the surface of the nanorod based on the localization of the fluorescent labels. Using the dipolar emission model for the luminescence allows for the retention of more emission events from the fluorescent label, after applying a fitting threshold, and yields a more robust reconstructed image containing more centroid points that show the apparent locations of the dsDNA. Unfortunately, the sizes of the reconstructed nanorod images were smaller than expected, despite the use of the more accurate model for the gold luminescence, suggesting that the photophysics of this coupled dye–nanorod system are more complicated than when using the isolated fluorophores.

This Article introduces surface-enhanced Raman scattering (SERS) orientational imaging as a powerful far-field optical technique for determining the in-plane and out-of-plane orientations of SERS-active nanoparticle dimers. Optical images of Rhodamine 6G (R6G) SERS emission patterns are measured and correlated with atomic force microscopy (AFM) images of the associated SERS-active silver nanoparticle dimers. The AFM is used to measure individual silver nanoparticle dimer orientations and height asymmetry, defining in-plane and out-of-plane angles associated with the dimer geometry. Theoretical emission pattern images based on these angles are generated using a simple dipole emission model and show excellent agreement with the experimental emission patterns. This technique provides a rapid all-optical technique to analyze the orientation of SERS active nanoparticle dimers.