We report a study of the shape-dependent spectral response of the gold nanoparticle surface plasmon resonance at various electron densities to provide mechanistic insight into the role of capacitive charging, a topic of some debate. We demonstrate a morphology-dependent spectral response for gold nanoparticles due to capacitive charging using single-particle spectroscopy in an inert electrochemical environment. A decrease in plasmon energy and increase in spectral width for gold nanospheres and nanorods was observed as the electron density was tuned through a potential window of −0.3 to 0.1 V. The combined observations could not be explained by existing theories. A new quantum theory for charging based on the random phase approximation was developed. Additionally, the redox reaction of gold oxide formation was probed using single-particle plasmon voltammetry to reproduce the reduction peak from the bulk cyclic voltammetry. These results deepen our understanding of the relationship between optical and electronic properties in plasmonic nanoparticles and provide insight toward their potential applications in directed electrocatalysis.

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.

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.

Interactions between fluorophores and plasmonic nanoparticles modify the fluorescence intensity, shape, and position of the observed emission pattern, thus inhibiting efforts to optically super-resolve plasmonic nanoparticles. Herein, we investigate the accuracy of localizing dye fluorescence as a function of the spectral and spatial separations between fluorophores (Alexa 647) and gold nanorods (NRs). The distance at which Alexa 647 interacts with NRs is varied by layer-by-layer polyelectrolyte deposition while the spectral separation is tuned by using NRs with varying localized surface plasmon resonance (LSPR) maxima. For resonantly coupled Alexa 647 and NRs, emission to the far field through the NR plasmon is highly prominent, resulting in underestimation of NR sizes. However, we demonstrate that it is possible to improve the accuracy of the emission localization when both the spectral and spatial separations between Alexa 647 and the LSPR are optimized.

Nanoparticle and thin film surface plasmons are highly sensitive to electrochemically induced dielectric changes. We exploited this sensitivity to detect reversible electrochemical potential-driven anion adsorption by developing single-particle plasmon voltammetry (spPV) using plasmonic nanoparticles. spPV was used to detect sulfate electroadsorption to individual Au nanoparticles. By comparing both semiconducting and metallic thin film substrates with Au nanoparticle monomers and dimers, we demonstrated that using Au film substrates improved the signal in detecting sulfate electroadsorption and desorption through adsorbate modulated thin film conductance. Using single-particle surface plasmon spectroscopic techniques, we constructed spPV to sense sulfate, acetate, and perchlorate adsorption on coupled Au nanoparticles. spPV extends dynamic spectroelectrochemical sensing to the single-nanoparticle level using both individual plasmon resonance modes and total scattering intensity fluctuations.

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.

Functionalization of polymer films with ion exchange ligands is a common method for creating surfaces optimized for separations and purification. Surfaces are typically evaluated for their ability to retain target molecules, but this retention encompasses a variety of physical and chemical processes. In this work we use single molecule fluorescence microscopy to investigate two ion exchange ligands that enhance surface binding of their respective target proteins. Single molecule tracking reveals that in addition to increasing the rate of surface interaction, functionalization can also increase the surface mobility of the target molecules resulting in large areas of the membrane being explored during adsorption, likely due to hopping of the protein molecules to adjacent binding sites. Hopping was only observed for one of the ligands and not the other. The enhanced mobility was found to be proportional to the UV exposure time during ligand grafting, which suggests that the hopping scales with the grafted polymer chain length.

A spectroelectrochemical flow cell is used to probe the localized surface plasmon resonance (LSPR) of the same single gold nanorods (AuNRs) in sodium fluoride, sodium chloride, and sodium bromide electrolytes using dark-field scattering microscopy. The changes in resonance energy, line width (full-width at half-maximum, fwhm), and peak intensity of a Lorentzian fit to single AuNR scattering spectra as the rods are charged are compared to determine the role of anion adsorption. We demonstrate that at positive potentials up to +0.25 V relative to a Pt quasi-reference electrode, the induced changes in the LSPR are independent of halide anion. At more positive potentials (+0.3 to +0.35 V) bromide and chloride ions damp the AuNR LSPR, observed as an increase in the line width. At the most positive potential investigated in all three electrolyte solutions (+0.35 V), the AuNR scattering intensity decreases irreversibly in bromide electrolyte, indicating dissolution. The kinetics of the bromide-mediated dissolution can be controlled by the electrolyte concentration and show that the change in resonance energy due to dissolution increases with each cycle from negative to positive potential.

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.Keywords: correlation spectroscopy; nanorods; protein corona; superlocalization microscopy; surface plasmon;

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.

•We studied D17.4 aptamer/IgE association by ensemble and single-molecule methods.•Changes in activation enthalpy and entropy with NaCl concentration are small.•The C19A mutant of D17.4 showed slower dissociation kinetics than D17.4.•Single-molecule methods revealed heterogeneity not observable by ensemble methods.BackgroundThe IgE-binding DNA aptamer 17.4 is known to inhibit the interaction of IgE with the high-affinity IgE Fc receptor FcεRI. While this and other aptamers have been widely used and studied, there has been relatively little investigation of the kinetics and energetics of their interactions with their targets, by either single-molecule or ensemble methods.MethodsThe dissociation kinetics of the D17.4/IgE complex and the effects of temperature and ionic strength were studied using fluorescence anisotropy and single-molecule spectroscopy, and activation parameters calculated.ResultsThe dissociation of D17.4/IgE complex showed a strong dependence on temperature and salt concentration. The koff of D17.4/IgE complex was calculated to be (2.92 ± 0.18) × 10−3 s−1 at 50 mM NaCl, and (1.44 ± 0.02) × 10−2 s−1 at 300 mM NaCl, both in 1 mM MgCl2 and 25 °C. The dissociation activation energy for the D17.4/IgE complex, Ea, was 16.0 ± 1.9 kcal mol−1 at 50 mM NaCl and 1 mM MgCl2. Interestingly, we found that the C19A mutant of D17.4 with stabilized stem structure showed slower dissociation kinetics compared to D17.4. Single-molecule observations of surface-immobilized D17.4/IgE showed much faster dissociation kinetics, and heterogeneity not observable by ensemble techniques.ConclusionsThe increasing koff value with increasing salt concentration is attributed to the electrostatic interactions between D17.4/IgE. We found that both the changes in activation enthalpy and activation entropy are insignificant with increasing NaCl concentration. The slower dissociation of the mutant C19A/IgE complex is likely due to the enhanced stability of the aptamer.General significanceThe activation parameters obtained by applying transition state analysis to kinetic data can provide details on mechanisms of molecular recognition and have applications in drug design. Single-molecule dissociation kinetics showed greater kinetic complexity than was observed in the ensemble in-solution systems, potentially reflecting conformational heterogeneity of the aptamer. This article is part of a Special Issue entitled: Physiological Enzymology and Protein Functions.

Photoluminescent Au nanoparticles are appealing for biosensing and bioimaging applications because of their non-photobleaching and non-photoblinking emission. The mechanism of one-photon photoluminescence from plasmonic nanostructures is still heavily debated though. Here, we report on the one-photon photoluminescence of strongly coupled 50 nm Au nanosphere dimers, the simplest plasmonic molecule. We observe emission from coupled plasmonic modes as revealed by single-particle photoluminescence spectra in comparison to correlated dark-field scattering spectroscopy. The photoluminescence quantum yield of the dimers is found to be surprisingly similar to the constituent monomers, suggesting that the increased local electric field of the dimer plays a minor role, in contradiction to several proposed mechanisms. Aided by electromagnetic simulations of scattering and absorption spectra, we conclude that our data are instead consistent with a multistep mechanism that involves the emission due to radiative decay of surface plasmons generated from excited electron–hole pairs following interband absorption.Keywords: gold nanoparticle; nanoparticle dimer; photoluminescence; plasmon coupling; surface plasmon;

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.Keywords: correlation; diffusion; fluorescence microscopy; hydrogel; liquid crystal; super-resolution;

•Direct observation of individual functional ion-exchange ligands at varying salt.•Elution curves were assembled by combining ensemble kinetics and stochastic theory.•Ionic strength reduces heterogeneity of active adsorption sites.•Electrostatic screening and steric availability within the agarose support play a role.•Results help interpret a large body of previous results.The retention and elution of proteins in ion-exchange chromatography is routinely controlled by adjusting the mobile phase salt concentration. It has repeatedly been observed, as judged from adsorption isotherms, that the apparent heterogeneity of adsorption is lower at more-eluting, higher ionic strength. Here, we present an investigation into the mechanism of this phenomenon using a single-molecule, super-resolution imaging technique called motion-blur Points Accumulation for Imaging in Nanoscale Topography (mbPAINT). We observed that the number of functional adsorption sites was smaller at high ionic strength and that these sites had reduced desorption kinetic heterogeneity, and thus narrower predicted elution profiles, for the anion-exchange adsorption of α-lactalbumin on an agarose-supported, clustered-charge ligand stationary phase. Explanations for the narrowing of the functional population such as inter-protein interactions and protein or support structural changes were investigated through kinetic analysis, circular dichroism spectroscopy, and microscopy of agarose microbeads, respectively. The results suggest the reduction of heterogeneity is due to both electrostatic screening between the protein and ligand and tuning the steric availability within the agarose support. Overall, we have shown that single molecule spectroscopy can aid in understanding the influence of ionic strength on the population of functional adsorbent sites participating in the ion-exchange chromatographic separation of proteins.

Single particle tracking (SPT) techniques provide a microscopic approach to probe in vivo and in vitro structure and reactions. Automatic analysis of SPT data with high efficiency and accuracy spurs the development of SPT algorithms. In this perspective, we review a range of available techniques used in SPT analysis programs. In addition, we present an example SPT program step-by-step to provide a guide so that researchers can use, modify, and/or write a SPT program for their own purposes.

We introduce a step transition and state identification (STaSI) method for piecewise constant single-molecule data with a newly derived minimum description length equation as the objective function. We detect the step transitions using the Student’s t test and group the segments into states by hierarchical clustering. The optimum number of states is determined based on the minimum description length equation. This method provides comprehensive, objective analysis of multiple traces requiring few user inputs about the underlying physical models and is faster and more precise in determining the number of states than established and cutting-edge methods for single-molecule data analysis. Perhaps most importantly, the method does not require either time-tagged photon counting or photon counting in general and thus can be applied to a broad range of experimental setups and analytes.Keywords: change point; minimum description length; piecewise constant signal; state identification; step detection;

Chromatographic protein separations, immunoassays, and biosensing all typically involve the adsorption of proteins to surfaces decorated with charged, hydrophobic, or affinity ligands. Despite increasingly widespread use throughout the pharmaceutical industry, mechanistic detail about the interactions of proteins with individual chromatographic adsorbent sites is available only via inference from ensemble measurements such as binding isotherms, calorimetry, and chromatography. In this work, we present the direct superresolution mapping and kinetic characterization of functional sites on ion-exchange ligands based on agarose, a support matrix routinely used in protein chromatography. By quantifying the interactions of single proteins with individual charged ligands, we demonstrate that clusters of charges are necessary to create detectable adsorption sites and that even chemically identical ligands create adsorption sites of varying kinetic properties that depend on steric availability at the interface. Additionally, we relate experimental results to the stochastic theory of chromatography. Simulated elution profiles calculated from the molecular-scale data suggest that, if it were possible to engineer uniform optimal interactions into ion-exchange systems, separation efficiencies could be improved by as much as a factor of five by deliberately exploiting clustered interactions that currently dominate the ion-exchange process only accidentally.

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.

A hyperspectral imaging method was developed that allowed the identification of heterogeneous plasmon response from 50 nm diameter gold colloidal particles on a conducting substrate in a transparent three-electrode spectroelectrochemical cell under non-Faradaic conditions. At cathodic potentials, we identified three distinct behaviors from different nanoparticles within the same sample: irreversible chemical reactions, reversible chemical reactions, and reversible charge density tuning. The irreversible reactions in particular would be difficult to discern in alternate methodologies. Additional heterogeneity was observed when single nanoparticles demonstrating reversible charge density tuning in the cathodic regime were measured dynamically in anodic potential ranges. Some nanoparticles that showed charge density tuning in the cathodic range also showed signs of an additional chemical tuning mechanism in the anodic range. The expected changes in nanoparticle free-electron density were modeled using a charge density-modified Drude dielectric function and Mie theory, a commonly used model in colloidal spectroelectrochemistry. Inconsistencies between experimental results and predictions of this common physical model were identified and highlighted. The broad range of responses on even a simple sample highlights the rich experimental and theoretical playgrounds that hyperspectral single-particle electrochemistry opens.

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 cm2/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.

We demonstrate the application of superlocalization microscopy to identify sequence-specific portions of single-stranded DNA (ssDNA) with sequence resolution of 50 nucleotides, corresponding to a spatial resolution of 30 nm. Super-resolution imaging was achieved using a variation of a single-molecule localization method, termed as “motion blur” point accumulation for imaging in nanoscale topography (mbPAINT). The target ssDNA molecules were immobilized on the substrate. Short, dye-labeled, and complementary ssDNA molecules stochastically bound to the target ssDNA, with repeated binding events allowing super-resolution. Sequence specificity was demonstrated via the use of a control, noncomplementary probe. The results support the possibility of employing relatively inexpensive short ssDNAs to identify gene sequence specificity with improved resolution in comparison to the existing methods.Keywords: biosensing; materials and biointerfaces; mbPAINT; optical mapping; single-stranded DNA; super-resolution microscopy;

We find that citrate-stabilized gold nanoparticles aggregate and precipitate in saline solutions below the NaCl concentration of many bodily fluids and blood plasma. Our experiments indicate that this is due to complexation of the citrate anions with Na+ cations in solution. A dramatically enhanced colloidal stability is achieved when bovine serum albumin is adsorbed to the gold nanoparticle surface, completely preventing nanoparticle aggregation under harsh environmental conditions where the NaCl concentration is well beyond the isotonic point. Furthermore, we explore the mechanism of the formation of this albumin “corona” and find that monolayer protein adsorption is most likely ruled by hydrophobic interactions. As for many nanotechnology-based biomedical and environmental applications, particle aggregation and sedimentation are undesirable and could substantially increase the risk of toxicological side effects; the formation of the BSA corona presented here provides a low-cost biocompatible strategy for nanoparticle stabilization and transport in highly ionic environments.Keywords: Bovine serum albumin; Correlation spectroscopy; Diffusion; Gold nanoparticles; Protein corona; Surface plasmon

The complete and reversible charge-selective sequestration of fluorophores by a weak polyelectrolyte brush, poly(2-(dimethylamino)ethylmethacrylate) (PDMAEMA) was demonstrated using fluorescence correlation spectroscopy (FCS). The chemistry and thickness of the weak polyelectrolyte PDMAEMA was tuned reversibly between neutral and cationic polymer forms. Thus, by switching the pH successively, the brush architecture was tuned to selectively trap and release anionic dye probes while continuously excluding cationic molecules. In addition, line-scan FCS was implemented and applied for the first time to a synthetic polymer system and used to identify a new, slower diffusion time on the order of seconds for the sequestered anionic probe under acidic conditions. These results, which quantify the selective sequestration properties of the PDMAEMA brush, are important because they enable a better understanding of transport in polymers and establish a spectroscopic means of evaluating materials with proposed applications in separations science, charge storage/release, and environmental remediation.

Two maximum likelihood estimation (MLE) methods were developed for optimizing the analysis of single-molecule trajectories that include phenomena such as experimental noise, photoblinking, photobleaching, and translation or rotation out of the collection plane. In particular, short, single-molecule trajectories with photoblinking were studied, and our method was compared to existing analytical techniques applied to simulated data. The optimal method for various experimental cases was established, and the optimized MLE method was applied to a real experimental system: single-molecule diffusion of fluorescent molecular machines known as nanocars.

Functional polymers have a wide variety of applications ranging from energy storage to drug delivery. For energy storage applications, desirable material properties include low cost, high charge storage and/or mobility, and low rates of degradation. Isotropic thin films have been used for many of these types of applications, but research suggests that different structures such as polymer brushes can improve charge transport by an order of magnitude. Supported polymer brush structures produced by “grafting-from” polymerization methods offer a framework for a controlled study of these materials on the molecular scale. Using these materials, researchers can study the basis of hindered diffusion because they contain a relatively homogeneous polyelectrolyte membrane. In addition, researchers can use fluorescent molecular probes with different charges to examine steric and Coulombic contributions to transport near and within polymer brushes.In this Account, we discuss recent progress in using fluorescence correlation spectroscopy, single-molecule polarization-resolved spectroscopy, and a novel three-dimensional orientational technique to understand the transport of charged dye probes interacting with the strong polyanionic brush, poly(styrene sulfonate). Our preliminary experiments demonstrate that a cationic dye, Rhodamine 6G, probes the brush as a counterion, and diffusion is therefore dominated by Coulombic forces, which results in a 10 000-fold decrease in the diffusion coefficient in comparison with free diffusion. We also support our experimental results with molecular dynamics simulations. Further experiments show that, up to 50% of the time, Rhodamine 6G translates within the brush without significant rotational diffusion, which indicates a strong deviation from Fickian transport mechanisms (in which translational and rotational diffusion are related directly through parameters such as chemical potential, size, solution viscosity, and thermal properties). To understand this oriented transport, we discuss the development of an experimental technique that allows us to quantify the three-dimensional orientation on the time scale of intrabrush transport. This method allowed us to identify a unique orientational transport direction for Rhodamine 6G within the poly(styrene sulfonate) brush and to report preliminary evidence for orientational dye “hopping”.

The resolution of complex interactions found in single-molecule fluorescence resonance energy transfer (smFRET) experiments is hindered by noise. Wavelet shrinkage is proven to reduce noise, but traditional methods introduce artifacts when acting on discontinuous signals, such as those acquired in smFRET experiments. Modifications to the basic method that are specific to smFRET are developed and tested on simulated systems. Use of the Haar wavelet basis produces the most optimally denoised estimates. We also assess various thresholding methods, develop a time-localized noise estimator, and implement a translation-invariant wavelet transformation to reduce artifacts associated with discontinuities and inadequate distinction of noise. The time-local estimator enhances noise reduction by 5−20%, and translation-invariant transformation nearly eliminates the aforementioned artifacts. Kinetic parameters extracted from denoised estimates are accurate to within 5% of the simulated values. Overall, the improved resolution results in the complete and accurate characterization of both simple and complex smFRET systems.

A strong intrinsic signal is advantageous over labeling for optical detection of nanoparticles. Intense scattering and absorption by the surface plasmon resonance, which exceeds molecular cross sections, provides a direct method for visualizing noble metal nanoparticles. While two-photon luminescence in gold nanoparticles yields a strong signal, one-photon luminescence is generally regarded to be much weaker and has seldom been employed for optical nanoparticle detection. In this article we investigated one-photon luminescence of gold nanospheres and nanorods using single particle spectroscopy with excitation at 514 and 633 nm. We characterized the polarization dependence, determined the quantum yield, and present a mechanism describing one-photon luminescence. Our results suggest fast interconversion between surface plasmons and hot electron–hole pairs and show that the luminescence occurs via emission by a surface plasmon. Using the information obtained from the single particle studies, we were able to successfully employ one-photon luminescence for correlation spectroscopy measurements and to correctly interpret auto- and cross-correlation functions, which were used to determine the hydrodynamic sizes of several gold nanoparticle samples and to extract rotational dynamics of nanorods. Because of the difference in size dependence for one-photon luminescence compared to scattering, luminescence correlation spectroscopy of metal nanoparticles is advantageous as it is not as strongly affected by the presence of larger nanoparticles or aggregates. This was verified by measuring luminescence as well as scattering correlation traces for a mixture of nanoparticles containing 98% 57 nm and 2% 96 nm gold nanospheres.

The present work reports on in situ observations of the interaction of organic dye probe molecules and dye-labeled protein with different poly(ethylene glycol) (PEG) architectures (linear, dendron, and bottle brush). Fluorescence correlation spectroscopy (FCS) and single molecule event analysis were used to examine the nature and extent of probe–PEG interactions. The data support a sieve-like model in which size-exclusion principles determine the extent of probe–PEG interactions. Small probes are trapped by more dense PEG architectures and large probes interact more with less dense PEG surfaces. These results, and the tunable pore structure of the PEG dendrons employed in this work, suggest the viability of electrochemically-active materials for tunable surfaces.Graphical abstractHighlights► Interactions of organic dye and dye-labeled protein with several PEG architectures. ► Linear, dendron, and bottle brush architectures studied. ► Sieve-like behavior of PEGs involving size-exclusion principles. ► Small probes trapped by dense PEG; large probes interact with sparse PEG. ► Dendrons allow variable PEG density, optimal for fabrication of tunable surfaces.

Single-molecule fluorescence spectroscopy is employed to reveal 3D details of the mechanisms underpinning ion transport in a polyelectrolyte thin film possessing polymer-brush nanoscale order. The ability to resolve fluorescence emission over three discrete polarization angles reveals that these ordered materials impart 3D orientation to charged, diffusing molecules. The experiments, supported by simulations, report global orientation parameters for molecular transport, track dipole angle progressions over time, and identify a unique transport mechanism: translational diffusion with restricted rotation. In general, realization of this experimental method for translational diffusion in systems exhibiting basic orientation should lend itself to evaluation of transport in a variety of important, ordered, functional materials.Keywords: diffusion; polymer brush; reorientation; single molecule; spectroscopy;

Fluorescence correlation spectroscopy and single molecule burst analysis were used to measure the effects of glass surface interactions on the diffusion of two common fluorescent dyes, one cationic and one anionic. The effects of dye−surface interactions on measured diffusion rates as a function of distance from the surface were investigated. Use of a three-axis piezo stage, combined with reference calibration measurements, enabled the accurate acquisition of surface-distance-dependent transport data. This analysis reveals attractive interactions between the cationic dye and the surface, which significantly alter the extracted diffusion values and persist in the measurements up to 1.0 μm from the surface. The Coulomb attraction between the cationic dye and the surface also results in rare, long-lived association events that lead to irreproducibility in extracted diffusion values. In addition to an assignment of the association lifetime for these events, this paper demonstrates that, if experiments must be performed with cationic probes near a glass surface, the use of solution electrolytes can eliminate deleterious dye−surface interactions, as the dyes were tested in a variety of environments. Finally, our data demonstrate that a better dye choice is an anionic probe, which exhibits no depth dependence of diffusion characteristics above a glass surface.

Single molecule polarization and fluorescence correlation spectroscopy were used to evaluate heterogeneous transport mechanisms of molecular ions within supported polyelectrolyte brushes. Modes of diffusive transport include periods of significantly restricted rotational motion, often maintained over tens of milliseconds; periods of fast molecular rotation; and occasional adsorption of fluorescent probe molecules in the brush. The studies reveal rapid switching between orientational states during each observed mode of motion. Through quantitative analysis of state occupation times, the rate constants for transitions from weakly associated to strongly associated states were extracted. Additionally, the pH dependence of the ion transport rates in the brush exhibits an abrupt, rather than continuous, trend. These single molecule studies demonstrate the presence of dynamic anisotropic interactions between the charged molecular probe and the polymer brush and provide experimental evidence of stimuli responsive switchable transport functionality in the polyelectrolyte brush.

The N-methyl-D-aspartate receptor (NMDAR) is a member of the glutamate receptor family of proteins and is responsible for excitatory transmission. Activation of the receptor is thought to be controlled by conformational changes in the ligand binding domain (LBD); however, glutamate receptor LBDs can occupy multiple conformations even in the activated form. This work probes equilibrium transitions among NMDAR LBD conformations by monitoring the distance across the glycine-bound LBD cleft using single-molecule Förster resonance energy transfer (smFRET). Recent improvements in photoprotection solutions allowed us to monitor transitions among the multiple conformations. Also, we applied a recently developed model-free algorithm called “step transition and state identification” to identify the number of states, their smFRET efficiencies, and their interstate kinetics. Reversible interstate conversions, corresponding to transitions among a wide range of cleft widths, were identified in the glycine-bound LBD, on much longer timescales compared to channel opening. These transitions were confirmed to be equilibrium in nature by shifting the distribution reversibly via denaturant. We found that the NMDAR LBD proceeds primarily from one adjacent smFRET state to the next under equilibrium conditions, consistent with a cleft-opening/closing mechanism. Overall, by analyzing the state-to-state transition dynamics and distributions, we achieve insight into specifics of long-lived LBD equilibrium structural dynamics, as well as obtain a more general description of equilibrium folding/unfolding in a conformationally dynamic protein. The relationship between such long-lived LBD dynamics and channel function in the full receptor remains an open and interesting question.

A method to denoise single-molecule fluorescence resonance energy (smFRET) trajectories using wavelet detail thresholding and Bayesian inference is presented. Bayesian methods are developed to identify fluorophore photoblinks in the time trajectories. Simulated data are used to quantify the improvement in static and dynamic data analysis. Application of the method to experimental smFRET data shows that it distinguishes photoblinks from large shifts in smFRET efficiency while maintaining the important advantage of an unbiased approach. Known sources of experimental noise are examined and quantified as a means to remove their contributions via soft thresholding of wavelet coefficients. A wavelet decomposition algorithm is described, and thresholds are produced through the knowledge of noise parameters in the discrete-time photon signals. Reconstruction of the signals from thresholded coefficients produces signals that contain noise arising only from unquantifiable parameters. The method is applied to simulated and observed smFRET data, and it is found that the denoised data retain their underlying dynamic properties, but with increased resolution.

Single particle tracking (SPT) techniques provide a microscopic approach to probe in vivo and in vitro structure and reactions. Automatic analysis of SPT data with high efficiency and accuracy spurs the development of SPT algorithms. In this perspective, we review a range of available techniques used in SPT analysis programs. In addition, we present an example SPT program step-by-step to provide a guide so that researchers can use, modify, and/or write a SPT program for their own purposes.