Light emission from plasmonic nanostructures.

The mechanism of light emission from metallic nanoparticles has been a subject of debate in recent years. Photoluminescence and electronic Raman scattering mechanisms have both been proposed to explain the observed emission from plasmonic nanostructures. Recent results from Stokes and anti-Stokes emission spectroscopy of single gold nanorods using continuous wave laser excitation carried out in our laboratory are summarized here. We show that varying excitation wavelength and power change the energy distribution of hot carriers and impact the emission spectral lineshape. We then examine the role of interband and intraband transitions in the emission lineshape by varying the particle size. We establish a relationship between the single particle emission quantum yield and its corresponding plasmonic resonance quality factor, which we also tune through nanorod crystallinity. Finally, based on anti-Stokes emission, we extract electron temperatures that further suggest a hot carrier based mechanism. The central role of hot carriers in our systematic study on gold nanorods as a model system supports a Purcell effect enhanced hot carrier photoluminescence mechanism. We end with a discussion on the impact of understanding the light emission mechanism on fields utilizing hot carrier distributions, such as photocatalysis and nanothermometry.

Plasmon Energy Transfer in Hybrid Nanoantennas.

Plasmonic metal nanoparticles exhibit large dipole moments upon photoexcitation and have the potential to induce electronic transitions in nearby materials, but fast internal relaxation has to date limited the spatial range and efficiency of plasmonic mediated processes. In this work, we use photo-electrochemistry to synthesize hybrid nanoantennas comprised of plasmonic nanoparticles with photoconductive polymer coatings. We demonstrate that the formation of the conductive polymer is selective to the nanoparticles and that polymerization is enhanced by photoexcitation. In situ spectroscopy and simulations support a mechanism in which up to 50% efficiency of nonradiative energy transfer is achieved. These hybrid nanoantennas combine the unmatched light-harvesting properties of a plasmonic antenna with the similarly unmatched device processability of a polymer shell.

Increased Intraband Transitions in Smaller Gold Nanorods Enhance Light Emission.

Photoinduced light emission from plasmonic nanoparticles has attracted considerable interest within the scientific community because of its potential applications in sensing, imaging, and nanothermometry. One of the suggested mechanisms for the light emission from plasmonic nanoparticles is the plasmon-enhanced radiative recombination of hot carriers through inter- and intraband transitions. Here, we investigate the nanoparticle size dependence on the photoluminescence through a systematic analysis of gold nanorods with similar aspect ratios. Using single-particle emission and scattering spectroscopy along with correlated scanning electron microscopy and electromagnetic simulations, we calculate the emission quantum yields and Purcell enhancement factors for individual gold nanorods. Our results show strong size-dependent quantum yields in gold nanorods, with higher quantum yields for smaller gold nanorods. Furthermore, by determining the relative contributions to the photoluminescence from inter- and intraband transitions, we deduce that the observed size dependence predominantly originates from the size dependence of intraband transitions. Specifically, within the framework of Fermi's golden rule for radiative recombination of excited charge carriers, we demonstrate that the Purcell factor enhancement alone cannot explain the emission size dependence and that changes in the transition matrix elements must also occur. Those changes are due to electric field confinement enhancing intraband transitions. These results provide vital insight into the intraband relaxation in metallic nanoconfined systems and therefore are of direct importance to the rapidly developing field of plasmonic photocatalysis.

Polymer Free Volume Effects on Protein Dynamics in Polystyrene Revealed by Single-Molecule Spectroscopy.

Protein-polymer interactions are critical to applications ranging from biomedical devices to chromatographic separations. The mechanistic relationship between the microstructure of polymer chains and protein interactions is challenging to quantify and not well studied. Here, single-molecule microscopy is used to compare the dynamics of two model proteins, α-lactalbumin and lysozyme, at the interface of uncharged polystyrene with varied molecular weights. The two proteins exhibit different surface interaction mechanisms despite having a similar size and structure. α-Lactalbumin exhibits interfacial adsorption-desorption with residence times that depend on polymer molecular weight. Lysozyme undergoes a continuous time random walk at the polystyrene surface with residence times that also depend on the molecular weight of polystyrene. Single-molecule observables suggest that the hindered continuous time random walk dynamics displayed by lysozyme are determined by the polystyrene free volume, a finding supported by thermal annealing and solvent quality studies. Hindered dynamics are dominated by short-range hydrophobic interactions where the contributions of electrostatic forces are negligible. This work establishes a relationship between the microscale structure (i.e., free volume) of polystyrene polymer chains to nanoscale interfacial protein dynamics.

Anti-Stokes Emission from Hot Carriers in Gold Nanorods.

The origin of light emission from plasmonic nanoparticles has been strongly debated lately. It is present as the background of surface-enhanced Raman scattering and, despite the low yield, has been used for novel sensing and imaging applications because of its photostability. Although the role of surface plasmons as an enhancing antenna is widely accepted, the main controversy regarding the mechanism of the emission is its assignment to either radiative recombination of hot carriers (photoluminescence) or electronic Raman scattering (inelastic light scattering). We have previously interpreted the Stokes-shifted emission from gold nanorods as the Purcell effect enhanced radiative recombination of hot carriers. Here we specifically focused on the anti-Stokes emission from single gold nanorods of varying aspect ratios with excitation wavelengths below and above the interband transition threshold while still employing continuous wave lasers. Analysis of the intensity ratios between Stokes and anti-Stokes emission yields temperatures that can only be interpreted as originating from the excited electron distribution and not a thermally equilibrated phonon population despite not using pulsed laser excitation. Consistent with this result as well as previous emission studies using ultrafast lasers, the power-dependence of the upconverted emission is nonlinear and gives the average number of participating photons as a function of emission wavelength. Our findings thus show that hot carriers and photoluminescence play a major role in the upconverted emission.

Polycrystallinity of Lithographically Fabricated Plasmonic Nanostructures Dominates Their Acoustic Vibrational Damping.

The study of acoustic vibrations in nanoparticles provides unique and unparalleled insight into their mechanical properties. Electron-beam lithography of nanostructures allows precise manipulation of their acoustic vibration frequencies through control of nanoscale morphology. However, the dissipation of acoustic vibrations in this important class of nanostructures has not yet been examined. Here we report, using single-particle ultrafast transient extinction spectroscopy, the intrinsic damping dynamics in lithographically fabricated plasmonic nanostructures. We find that in stark contrast to chemically synthesized, monocrystalline nanoparticles, acoustic energy dissipation in lithographically fabricated nanostructures is solely dominated by intrinsic damping. A quality factor of Q = 11.3 ± 2.5 is observed for all 147 nanostructures, regardless of size, geometry, frequency, surface adhesion, and mode. This result indicates that the complex Young's modulus of this material is independent of frequency with its imaginary component being approximately 11 times smaller than its real part. Substrate-mediated acoustic vibration damping is strongly suppressed, despite strong binding between the glass substrate and Au nanostructures. We anticipate that these results, characterizing the optomechanical properties of lithographically fabricated metal nanostructures, will help inform their design for applications such as photoacoustic imaging agents, high-frequency resonators, and ultrafast optical switches.

Photoluminescence of Gold Nanorods: Purcell Effect Enhanced Emission from Hot Carriers.

We demonstrate, experimentally and theoretically, that the photon emission from gold nanorods can be viewed as a Purcell effect enhanced radiative recombination of hot carriers. By correlating the single-particle photoluminescence spectra and quantum yields of gold nanorods measured for five different excitation wavelengths and varied excitation powers, we illustrate the effects of hot carrier distributions evolving through interband and intraband transitions and the photonic density of states on the nanorod photoluminescence. Our model, using only one fixed input parameter, describes quantitatively both emission from interband recombination and the main photoluminescence peak coinciding with the longitudinal surface plasmon resonance.

Variable Lysozyme Transport Dynamics on Oxidatively Functionalized Polystyrene Films.

Tuning protein adsorption dynamics at polymeric interfaces is of great interest to many biomedical and material applications. Functionalization of polymer surfaces is a common method to introduce application-specific surface chemistries to a polymer interface. In this work, single-molecule fluorescence microscopy is utilized to determine the adsorption dynamics of lysozyme, a well-studied antibacterial protein, at the interface of polystyrene oxidized via UV exposure and oxygen plasma and functionalized by ligand grafting to produce varying degrees of surface hydrophilicity, surface roughness, and induced oxygen content. Single-molecule tracking indicates lysozyme loading capacities, and surface mobility at the polymer interface is hindered as a result of all functionalization techniques. Adsorption dynamics of lysozyme depend on the extent and the specificity of the oxygen functionalities introduced to the polystyrene surface. Hindered adsorption and mobility are dominated by hydrophobic effects attributed to water hydration layer formation at the functionalized polystyrene surfaces.

Competitive multicomponent anion exchange adsorption of proteins at the single molecule level.

We use single molecule spectroscopy to study a multicomponent, competitive protein adsorption system. Fluorescently-labeled α-lactalbumin proteins are super-resolved adsorbing to cationic anion-exchange ligands in the presence of a competitor, insulin. We find that the competitor reduces the number of binding events by blocking ligands throughout the observed measurement time while the single-site adsorption kinetics are unchanged.

Single Particle Tracking: From Theory to Biophysical Applications.

After three decades of developments, single particle tracking (SPT) has become a powerful tool to interrogate dynamics in a range of materials including live cells and novel catalytic supports because of its ability to reveal dynamics in the structure-function relationships underlying the heterogeneous nature of such systems. In this review, we summarize the algorithms behind, and practical applications of, SPT. We first cover the theoretical background including particle identification, localization, and trajectory reconstruction. General instrumentation and recent developments to achieve two- and three-dimensional subdiffraction localization and SPT are discussed. We then highlight some applications of SPT to study various biological and synthetic materials systems. Finally, we provide our perspective regarding several directions for future advancements in the theory and application of SPT.

Super Temporal-Resolved Microscopy (STReM).

Super-resolution microscopy typically achieves high spatial resolution, but the temporal resolution remains low. We report super temporal-resolved microscopy (STReM) to improve the temporal resolution of 2D super-resolution microscopy by a factor of 20 compared to that of the traditional camera-limited frame rate. This is achieved by rotating a phase mask in the Fourier plane during data acquisition and then recovering the temporal information by fitting the point spread function (PSF) orientations. The feasibility of this technique is verified with both simulated and experimental 2D adsorption/desorption and 2D emitter transport. When STReM is applied to measure protein adsorption at a glass surface, previously unseen dynamics are revealed.

Single-Molecule Kinetics of Protein Adsorption on Thin Nylon-6,6 Films.

Understanding and controlling protein adsorption on surfaces is critical to a range of biological and materials applications. Kinetic details that provide the equilibrium and nonequilibrium mechanisms are difficult to acquire. In this work, single-molecule fluorescence microscopy was used to study the adsorption of Alexa 555 labeled α-lactalbumin (α-LA) on two chemically identical but morphologically different polymer surfaces: flat and porous nylon-6,6 thin films. The adsorption kinetics of spatially resolved single molecule α-LA binding to nylon films were quantified by a monolayer adsorption model. The surface morphology of the porous nylon-6,6 films increased the number of adsorption sites but decreased the binding affinity compared to the flat films. Such single-molecule based kinetic studies may be extended to various protein-polymer interactions.

Generalized recovery algorithm for 3D super-resolution microscopy using rotating point spread functions.

Super-resolution microscopy with phase masks is a promising technique for 3D imaging and tracking. Due to the complexity of the resultant point spread functions, generalized recovery algorithms are still missing. We introduce a 3D super-resolution recovery algorithm that works for a variety of phase masks generating 3D point spread functions. A fast deconvolution process generates initial guesses, which are further refined by least squares fitting. Overfitting is suppressed using a machine learning determined threshold. Preliminary results on experimental data show that our algorithm can be used to super-localize 3D adsorption events within a porous polymer film and is useful for evaluating potential phase masks. Finally, we demonstrate that parallel computation on graphics processing units can reduce the processing time required for 3D recovery. Simulations reveal that, through desktop parallelization, the ultimate limit of real-time processing is possible. Our program is the first open source recovery program for generalized 3D recovery using rotating point spread functions.

Adsorption and Unfolding of a Single Protein Triggers Nanoparticle Aggregation.

The response of living systems to nanoparticles is thought to depend on the protein corona, which forms shortly after exposure to physiological fluids and which is linked to a wide array of pathophysiologies. A mechanistic understanding of the dynamic interaction between proteins and nanoparticles and thus the biological fate of nanoparticles and associated proteins is, however, often missing mainly due to the inadequacies in current ensemble experimental approaches. Through the application of a variety of single molecule and single particle spectroscopic techniques in combination with ensemble level characterization tools, we identified different interaction pathways between gold nanorods and bovine serum albumin depending on the protein concentration. Overall, we found that local changes in protein concentration influence everything from cancer cell uptake to nanoparticle stability and even protein secondary structure. We envision that our findings and methods will lead to strategies to control the associated pathophysiology of nanoparticle exposure in vivo.

Characterization of Porous Materials by Fluorescence Correlation Spectroscopy Super-resolution Optical Fluctuation Imaging.

Porous materials such as cellular cytosol, hydrogels, and block copolymers have nanoscale features that determine macroscale properties. Characterizing the structure of nanopores is difficult with current techniques due to imaging, sample preparation, and computational challenges. We produce a super-resolution optical image that simultaneously characterizes the nanometer dimensions of and diffusion dynamics within porous structures by correlating stochastic fluctuations from diffusing fluorescent probes in the pores of the sample, dubbed here as "fluorescence correlation spectroscopy super-resolution optical fluctuation imaging" or "fcsSOFI". Simulations demonstrate that structural features and diffusion properties can be accurately obtained at sub-diffraction-limited resolution. We apply our technique to image agarose hydrogels and aqueous lyotropic liquid crystal gels. The heterogeneous pore resolution is improved by up to a factor of 2, and diffusion coefficients are accurately obtained through our method compared to diffraction-limited fluorescence imaging and single-particle tracking. Moreover, fcsSOFI allows for rapid and high-throughput characterization of porous materials. fcsSOFI could be applied to soft porous environments such hydrogels, polymers, and membranes in addition to hard materials such as zeolites and mesoporous silica.

Single-molecule FRET studies of HIV TAR-DNA hairpin unfolding dynamics.

We directly measure the dynamics of the HIV trans-activation response (TAR)-DNA hairpin with multiple loops using single-molecule Förster resonance energy transfer (smFRET) methods. Multiple FRET states are identified that correspond to intermediate melting states of the hairpin. The stability of each intermediate state is calculated from the smFRET data. The results indicate that hairpin unfolding obeys a "fraying and peeling" mechanism, and evidence for the collapse of the ends of the hairpin during folding is observed. These results suggest a possible biological function for hairpin loops serving as additional fraying centers to increase unfolding rates in otherwise stable systems. The experimental and analytical approaches developed in this article provide useful tools for studying the mechanism of multistate DNA hairpin dynamics and of other general systems with multiple parallel pathways of chemical reactions.

Charge-dependent transport switching of single molecular ions in a weak polyelectrolyte multilayer.

The tunable nature of weak polyelectrolyte multilayers makes them ideal candidates for drug loading and delivery, water filtration, and separations, yet the lateral transport of charged molecules in these systems remains largely unexplored at the single molecule level. We report the direct measurement of the charge-dependent, pH-tunable, multimodal interaction of single charged molecules with a weak polyelectrolyte multilayer thin film, a 10 bilayer film of poly(acrylic acid) and poly(allylamine hydrochloride) PAA/PAH. Using fluorescence microscopy and single-molecule tracking, two modes of interaction were detected: (1) adsorption, characterized by the molecule remaining immobilized in a subresolution region and (2) diffusion trajectories characteristic of hopping (D ∼ 10(-9) cm(2)/s). Radius of gyration evolution analysis and comparison with simulated trajectories confirmed the coexistence of the two transport modes in the same single molecule trajectories. A mechanistic explanation for the probe and condition mediated dynamics is proposed based on a combination of electrostatics and a reversible, pH-induced alteration of the nanoscopic structure of the film. Our results are in good agreement with ensemble studies conducted on similar films, confirm a previously-unobserved hopping mechanism for charged molecules in polyelectrolyte multilayers, and demonstrate that single molecule spectroscopy can offer mechanistic insight into the role of electrostatics and nanoscale tunability of transport in weak polyelectrolyte multilayers.

Photobleaching lifetimes of cyanine fluorophores used for single-molecule Förster resonance energy transfer in the presence of various photoprotection systems.

Lengthening smFRET lifetimes: We investigated various photoprotection system combinations to find the combination that optimally extended the photobleach lifetime of a Cy3/Cy5 smFRET pair attached to a DNA hairpin in a single-molecule environment. We found that the glucose/glucose oxygen-scavenging solution in combination with redox-based photostabilization solutions yielded the longest average photobleaching lifetimes.