Visualizing and Calculating Tip-Substrate Distance in Nanoscale Scanning Electrochemical Microscopy Using 3-Dimensional Super-Resolution Optical Imaging.

We report a strategy for the optical determination of tip-substrate distance in nanoscale scanning electrochemical microscopy (SECM) using three-dimensional super-resolution fluorescence imaging. A phase mask is placed in the emission path of our dual SECM/optical microscope, generating a double helix point spread function at the image plane, which allows us to measure the height of emitting objects relative to the focus of the microscope. By exciting both a fluorogenic reaction at the nanoscale electrode tip as well as fluorescent nanoparticles at the substrate, we are able to calculate the tip-substrate distance as the tip approaches the surface with precision better than 25 nm. Attachment of a fluorescent particle to the insulating sheath of the SECM tip extends this technique to nonfluorogenic electrochemical reactions. Correlated electrochemical and optical determination of tip-substrate distance yielded excellent agreement between the two techniques. Not only does super-resolution imaging offer a secondary feedback mechanism for measuring the tip-sample gap during SECM experiments, it also enables facile tip alignment and a strategy for accounting for electrode tilt relative to the substrate.

Observation of nanometer-sized electro-active defects in insulating layers by fluorescence microscopy and electrochemistry.

We report a method to study electro-active defects in passivated electrodes. This method couples fluorescence microscopy and electrochemistry to localize and size electro-active defects. The method was validated by comparison with a scanning probe technique, scanning electrochemical microscopy. We used our method for studying electro-active defects in thin TiO2 layers electrodeposited on 25 µm diameter Pt ultramicroelectrodes (UMEs). The permeability of the TiO2 layer was estimated by measuring the oxidation of ferrocenemethanol at the UME. Blocking of current ranging from 91.4 to 99.8% was achieved. Electro-active defects with an average radius ranging between 9 and 90 nm were observed in these TiO2 blocking layers. The distribution of electro-active defects over the TiO2 layer is highly inhomogeneous and the number of electro-active defect increases for lower degree of current blocking. The interest of the proposed technique is the possibility to quickly (less than 15 min) image samples as large as several hundreds of µm(2) while being able to detect electro-active defects of only a few tens of nm in radius.

Objective-Induced Point Spread Function Aberrations and Their Impact on Super-Resolution Microscopy.

This study demonstrates how different microscope objectives can lead to asymmetric imaging aberrations in the point spread function of dipolar emitters, which can adversely affect the quality of fit in super-resolution imaging. Luminescence from gold nanorods was imaged with four different objectives to measure the diffraction-limited emission and characterize deviations from the expected dipolar emission patterns. Each luminescence image was fit to a three-dipole emission model to generate fit residuals that visually relay aberrations in the point spread function caused by the different microscope objectives. Output parameters from the fit model were compared to experimentally measured values, and we find that while some objectives provide high quality fits across all nanorods studied, others show significant aberrations and are inappropriate for super-resolution imaging. This work presents a simple and robust strategy for quickly assessing the quality of point spread functions produced by different microscope objectives.

Triplet-state-mediated super-resolution imaging of fluorophore-labeled gold nanorods.

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.

Superlocalization surface-enhanced Raman scattering microscopy: comparing point spread function models in the ensemble and single-molecule limits.

In this report, we compare the effectiveness of various dipole and Gaussian point spread function (PSF) models for fitting diffraction-limited surface-enhanced Raman scattering (SERS) emission images from rhodamine 6G-labeled nanoparticle dimers at both the high-concentration and single-molecule limit. Of all models tested, a 3-axis dipole PSF gives the best approximation to the experimental PSF, although none of the models utilized in the study were without systematic error when fitting the experimental data. In the high-concentration regime, all models localize the SERS emission to a stationary centroid position, with the dipole models providing additional orientation parameters that closely match the geometry of the dimer, indicating that the molecules are coupled to all resonant plasmon modes of the nanostructure. In the single-molecule case, the different models show a mobile SERS centroid, consistent with single-molecule motion on the surface, but the behavior of the centroid is model-dependent. Despite the centroid mobility in the single-molecule regime, the dipole PSF models still give accurate orientation information on the underlying dimer structure, although with less precision than the ensemble-averaged samples.

Accuracy of superlocalization imaging using Gaussian and dipole emission point-spread functions for modeling gold nanorod luminescence.

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.

Super-resolution SERS imaging beyond the single-molecule limit: an isotope-edited approach.

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.

Discriminating nanoparticle dimers from higher order aggregates through wavelength-dependent SERS orientational imaging.

Surface-enhanced Raman scattering (SERS) orientational imaging is a recently developed all-optical technique able to determine SERS-active silver nanoparticle dimer orientations by observing lobe positions in SERS emission patterns formed by the directional polarization of SERS along the longitudinal axis of the dimer. Here we extend this technique to discriminate nanoparticle dimers from higher order aggregates by observing the wavelength dependence of SERS emission patterns, which are unchanged in nanoparticle dimers but show differences in higher order aggregates involving two or more nanoparticle junctions. The ability of SERS orientational imaging to identify stacked nanoparticles in higher order aggregates is also demonstrated. The shape of the SERS emission patterns originating from trimers labeled with low and high concentrations of dye is investigated, showing that the emission pattern lobes become less defined as the dye concentration increases. Dynamic fluctuations in the SERS emission pattern lobes are observed in aggregates labeled with low dye concentrations, as molecules diffuse into regions of higher electromagnetic enhancement in multiple nanoparticle junctions.

Subdiffraction-limited far-field Raman spectroscopy of single carbon nanotubes: an unenhanced approach.

We present a new approach for subdiffraction-limited far-field Raman spectroscopy of single carbon nanotubes using through-the-objective total internal reflection (TIR) excitation coupled to an atomic force microscope (AFM). By using this approach, we are able to detect spectroscopic signatures of structural changes along a single nanotube with nanometer resolution. A single multiwalled carbon nanotube is mounted on an AFM tip and imaged while tapping on the surface of a glass coverslip. As the angle of incidence of the excitation field is changed, we are able to tune the penetration depth of the evanescent field by steps as small as 2-10 nm. An increase in the ratio of the Raman D band (the disorder band) to G band (the in-plane graphitic band) of the carbon nanotube was demonstrated as the penetration depth decreased, indicating that most defects are concentrated at the end of the nanotube. We also observed frequency shifts of the G band as we changed the penetration depth. By changing the polarization of the incident beam, we are able detect the orientation and possible local curvature in the nanotubes. Coupling through-the-objective TIR with AFM is a powerful technique for studying structural and chemical properties of carbon nanotubes and can be easily extended to many other nanoscale/molecular systems.