Single Nanoparticle Tracking Research Articles

Nanomotor-enhanced transport of passive Brownian particles in porous media.

Artificial micro/nanomotors are expected to perform tasks in interface-rich and species-rich environments for biomedical and environmental applications. In these highly confined and interconnected pore spaces, active species may influence the motion of coexisting passive participants in unexpected ways. Using three-dimensional super-resolution single-nanoparticle tracking, we observed enhanced motion of passive nanoparticles due to the presence of dilute well-separated nanomotors in an interconnected pore space. This enhancement acted at distances that are large compared to the sizes of the particles and cavities, in contrast with the insignificant effect on the passive particles with the same dilute concentration of nanomotors in an unconfined liquid. Experiments and simulations suggested an amplification of hydrodynamic coupling between self-propelled and passive nanoparticles in the interconnected confined environment, which enhanced the effective energy for passive particles to escape cavities through small holes. This finding represents an emergent behavior of confined nanomotors and suggests new strategies for the development of antifouling membranes and drug delivery systems.

Label-free image scanning microscopy for kHz super-resolution imaging and single particle tracking.

We report the modification of a label-free image scanning microscope (ISM) to perform asynchronous 2D imaging at up to 24kHz while keeping the lateral resolution gain and background rejection of a regular label-free ISM setup. Our method uses a resonant mirror oscillating at 12kHz for one-direction scanning and a chromatic line for instantaneous scanning in the other direction. We adapt optical photon reassignment in this scanning regime to perform fully optical super-resolution imaging. We exploit the kHz imaging capabilities of this confocal imaging system for single nanoparticle tracking down to 20 nm for gold and 50 nm for silica particles as well as imaging freely moving Lactobacillus with improved resolution.

Direct Observation of Nanotracer Transport in Swarming Bacteria during Antibiotic Adaptation.

Microorganisms inevitably encounter environmental variations and thus need to develop necessary strategies to adjust the colonies for survival. Here, we use cooperating Serratia marcescens bacteria to reveal how the whole population responds to a gradually deteriorating habitat. When subjected to antibiotics with increasing doses, the swarming bacteria transform weak homogeneous turbulent flows to nematic jet flows with defects and vortices on a large scale, by which bacteria exploit these coherent flows to transfer material and/or information. We elucidate a complete view of detailed spatiotemporal transport behavior in such microscale active turbulence with single-nanoparticle tracking. The nanoparticles in these active flows are brought into the state with the up limit of superdiffusion by the bacterial collective response to the stronger antibiotic stimulation. Strikingly, we found that, under the strengthening stimulation from antibiotics, bacteria with only a small fraction of their community get elongated and facilitate the drastic turbulence transition and an enhanced superdiffusion. These findings imply a possible collective response mechanism against environmental variations.

Nanoscale and functional heterogeneity of the hippocampal extracellular space

The extracellular space (ECS) and its constituents play a crucial role in brain development, plasticity, circadian rhythm, and behavior, as well as brain diseases. Yet, since this compartment has an intricate geometry and nanoscale dimensions, its detailed exploration in live tissue has remained an unmet challenge. Here, we used a combination of single-nanoparticle tracking and super-resolution microscopy approaches to map the nanoscale dimensions of the ECS across the rodent hippocampus. We report that these dimensions are heterogeneous between hippocampal areas. Notably, stratum radiatum CA1 and CA3 ECS differ in several characteristics, a difference that gets abolished after digestion of the extracellular matrix. The dynamics of extracellular immunoglobulins vary within these areas, consistent with their distinct ECS characteristics. Altogether, we demonstrate that ECS nanoscale anatomy and diffusion properties are widely heterogeneous across hippocampal areas, impacting the dynamics and distribution of extracellular molecules.

Real-time six-dimensional spatiotemporal tracking of single anisotropic nanoparticles in live cells by integrated multifunctional light-sheet nanoscopy

An integrated multifunctional light-sheet nanoscopy (iMLSN) combined with differential interference contrast, total internal reflection, epifluorescence, a super-resolution radial fluctuation-stream module, and a wavelength-dependent light sheet was developed to simultaneously realize the six-dimensional (6D) vector-valued (three coordinates + rotational dynamics (azimuth and elevation angles) + transport speed) tracking of anisotropic nanoparticles in single living cells. The wavelength-dependent asymmetric scattering of light by gold nanorods was used to trigger signals depending on the polarizer angle, and real-time photo-switching was achieved by turning the polarizer, obtaining a series of super-resolution images, and tracking using different polarization directions and two channels. This technique was employed to directly observe native gold nanorods (AuNRs; 5 nm diameter × 15 nm length) and surface-functionalized AuNRs during their endocytosis and transport at the upper and attaching side membrane regions of single living cells, revealing that the AuNRs bound to the membrane receptors. The nanorods were subsequently internalized and transported away from the original entry spots. Detailed dynamic information regarding the rotation properties and endocytosis speed during the transmembrane process was also acquired for each region. The developed technique can be considered useful for the real-time monitoring of intracellular transport at various regions in single living cells, as well as for 6D vector-valued non-fluorescence super-resolution imaging and tracking.Graphical

Locally Structured On-Chip Optofluidic Hollow-Core Light Cages for Single Nanoparticle Tracking.

Nanoparticle tracking analysis (NTA) is a widely used methodology to investigate nanoscale systems at the single species level. Here, we introduce the locally structured on-chip optofluidic hollow-core light cage, as a novel platform for waveguide-assisted NTA. This hollow waveguide guides light by the antiresonant effect in a sparse array of dielectric strands and includes a local modification to realize aberration-free tracking of individual nano-objects, defining a novel on-chip solution with properties specifically tailored for NTA. The key features of our system are (i) well-controlled nano-object illumination through the waveguide mode, (ii) diffraction-limited and aberration-free imaging at the observation site, and (iii) a high level of integration, achieved by on-chip interfacing to fibers. The present study covers all aspects relevant for NTA including design, simulation, implementation via 3D nanoprinting, and optical characterization. The capabilities of the approach to precisely characterize practically relevant nanosystems have been demonstrated by measuring the solvency-induced collapse of a nanoparticle system which includes polymer brush-based shells that react to changes in the liquid environment. Our study unlocks the advantages of the light cage approach in the context of NTA, suggesting its application in various areas such as bioanalytics, life science, environmental science, or nanoscale material science in general.

United under stress: High-speed transport network emerging at bacterial living edge

Individuals tend to move freely when there is enough room but would act collectively for their survival under external stress. In the case of living cells, for instance, when a drop of low-density flagellated bacterial solution is transferred onto the agar surface, the initially disordered movement of individual bacteria would be replaced with coordinated cell swarming after a lag phase of a few hours. Here, we study how such cooperation is established while overcoming the disorder at the onset of the lag phase with single nanoparticle tracking. Upon the spreading of the droplet, the bacteria in the solution cluster and align near the almost immobilized contact line confining the drop, forming a narrow ring of cells. As individual cells move in and out of the ring continuously, certain flow patterns emerge in the inter-bacterial fluid. We reveal high-speed long-distance unidirectional flows with definite chirality along the outside of the ring, along the inside of the ring and across the ring. We speculate that these flows enable the fast and efficient transport, facilitating the communication and unification of the bacterial community.

Label free imaging and deep tracking of single biological nanoparticles in free solution by reflection enhanced dark field scattering microscopy

Label free optical imaging of single biological nanoparticles and deep tracking of their behaviors in free solution are very important in cell biology, but quite challenging due to the weak optical signals produced from small biological nanoparticles and the lack of deep tracking method in Z axis. Here, by simply introducing a reflective surface, we are able to significantly enhance the sensitivity of dark field scattering microscopy (DFSM). The significant enhancement on the sensitivity facilities the location of nanoparticles in Z axis over several hundred micrometers, enabling the deep tracking of single exosomes in free solution, as well as the clearly observation and identification of organelles at single cells with least exogenous interferences. The reflection enhanced dark field scattering microscopy (REDFSM) can be performed on a reflected dark field microscope without the reform of the equipment or the use of high power laser, implying its potential wide application in cell research, due to its improved sensitivity and simplicity. Compared with the widely used fluorescence microscopy which only sees what is labeled, the label free REDFSM can see most of the organelles in cell with high imaging quality, providing additional opportunity to discover new cell biological phenomena.

Alteration of NMDA receptor trafficking as a cellular hallmark of psychosis

A dysfunction of the glutamatergic transmission, especially of the NMDA receptor (NMDAR), constitutes one of the main biological substrate of psychotic disorders, such as schizophrenia. The NMDAR signaling hypofunction, through genetic and/or environmental insults, would cause a neurodevelopmental myriad of molecular, cellular, and network alterations that persist throughout life. Yet, the mechanisms underpinning NMDAR dysfunctions remain elusive. Here, we compared the membrane trafficking of NMDAR in three gold-standard models of schizophrenia, i.e., patient’s cerebrospinal fluids, genetic manipulations of susceptibility genes, and prenatal developmental alterations. Using a combination of single nanoparticle tracking, electrophysiological, biochemical, and behavioral approaches in rodents, we identified that the NMDAR trafficking in hippocampal neurons was consistently altered in all these different models. Artificial manipulations of the NMDAR surface dynamics with competing ligands or antibody-induced receptor cross-link in the developing rat brain were sufficient to regulate the adult acoustic startle reflex and compensate for an early pathological challenge. Collectively, we show that the NMDAR trafficking is markedly altered in all clinically relevant models of psychosis, opening new avenues of therapeutical strategies.

Characterization of the Functional Cross-Talk between Surface GABAA and Dopamine D5 Receptors.

The γ-aminobutyric acid type A receptor (GABAAR) plays a major role in fast inhibitory synaptic transmission and is highly regulated by the neuromodulator dopamine. In this aspect, most of the attention has been focused on the classical intracellular signaling cascades following dopamine G-protein-coupled receptor activation. Interestingly, the GABAAR and dopamine D5 receptor (D5R) have been shown to physically interact in the hippocampus, but whether a functional cross-talk occurs is still debated. In the present study, we use a combination of imaging and single nanoparticle tracking in live hippocampal neurons to provide evidence that GABAARs and D5Rs form dynamic surface clusters. Disrupting the GABAAR–D5R interaction with a competing peptide leads to an increase in the diffusion coefficient and the explored area of both receptors, and a drop in immobile synaptic GABAARs. By means of patch-clamp recordings, we show that this fast lateral redistribution of surface GABAARs correlates with a robust depression in the evoked GABAergic currents. Strikingly, it also shifts in time the expression of long-term potentiation at glutamatergic synapses. Together, our data both set the plasma membrane as the primary stage of a functional interplay between GABAAR and D5R, and uncover a non-canonical role in regulating synaptic transmission.

Unveiling Cellular Internalization Dynamics of Single Gold Nanorods by Tracking Their Orientational and Translational Motions

Single nanoparticle tracking (SPT) is a unique and powerful tool to investigate the interaction between nanoparticles and cells, which is of considerable importance for nanotechnology applications ...

Anomalous nanoparticle surface diffusion in LCTEM is revealed by deep learning-assisted analysis

The motion of nanoparticles near surfaces is of fundamental importance in physics, biology, and chemistry. Liquid cell transmission electron microscopy (LCTEM) is a promising technique for studying motion of nanoparticles with high spatial resolution. Yet, the lack of understanding of how the electron beam of the microscope affects the particle motion has held back advancement in using LCTEM for in situ single nanoparticle and macromolecule tracking at interfaces. Here, we experimentally studied the motion of a model system of gold nanoparticles dispersed in water and moving adjacent to the silicon nitride membrane of a commercial LC in a broad range of electron beam dose rates. We find that the nanoparticles exhibit anomalous diffusive behavior modulated by the electron beam dose rate. We characterized the anomalous diffusion of nanoparticles in LCTEM using a convolutional deep neural-network model and canonical statistical tests. The results demonstrate that the nanoparticle motion is governed by fractional Brownian motion at low dose rates, resembling diffusion in a viscoelastic medium, and continuous-time random walk at high dose rates, resembling diffusion on an energy landscape with pinning sites. Both behaviors can be explained by the presence of silanol molecular species on the surface of the silicon nitride membrane and the ionic species in solution formed by radiolysis of water in presence of the electron beam.

Real-Time Study of Protein Phase Separation with Spatiotemporal Analysis of Single-Nanoparticle Trajectories.

Liquid-liquid phase separation (LLPS) underlies the formation mechanism of membraneless biomolecular condensates locally to perform important physiological functions such as selective autophagy, but little is known about the relationship between their dynamic structural organization and biophysical properties. Here, a dark-field microscopy based single plasmonic nanoparticle tracking (DFSPT) technique was introduced to simultaneously monitor the diffusion dynamics of multiple gold nanorod (AuNR) probes in a protein LLPS system and to quantitatively characterize the spatiotemporal heterogeneity of the LLPS condensates during their phase transformation. Based on spatially and temporally resolved analysis of the diffusional behavior of the AuNRs, structure and material properties of p62 condensates, such as the viscoelasticity, the compartmentalization, and the recruitment of protein-covered nanoparticles into the large droplet, have been observed. Moreover, the nonsmooth droplet interface, its solidification after further phase transition or maturation, and the size effect of the inner vacuoles have also been revealed. Our method can be potentially applied to in vitro investigation of different reconstituted membrane-free biomolecular condensates and in vivo study of their dynamic evolution.

Nano-corrugated Nanochannels for In Situ Tracking of Single-Nanoparticle Translocation Dynamics

Dynamic motions of materials in liquid present a wealth of information concerning their physical properties. While fluorescence microscopy has been widely utilized for single-particle observations, the method cannot be used for characterizing fast motions of nanoscale objects due to the limited spatiotemporal resolution. Here, we report on a nanostructure strategy for nanoscale tracking of single nanoparticles. We fabricated a straight conduit in a SiO2 layer on a Si wafer with lithographically defined 30 nm-sized protrusions formed on the side walls. We performed resistive pulse measurements at a 1 MHz sampling rate wherein we found n-stepped current traces signifying n number of nanoparticles moving concurrently inside the nanochannel. Ensemble average of the ionic current signals revealed a peculiar feature reflecting the slightly stronger ion blockage at the nanoconstrictions between the protrusions, thereby proving the ability of nano-corrugation as physical gates to signify the precise positions of objects inside the nanofluidic channel. This in situ tracking approach elucidated steady-state motions of the nanoparticles moving at a constant speed under the counter-balanced electrophoretic and viscous drag forces, which also allowed estimations of their surface charge densities. The present method can be utilized as a speedometer for nanoscale objects of virtually any size as long as they are able to be put through the sensing zones with potential applications for single-molecule time-of-flight mass spectrometry.

Long-distance Transport in Bacterial Swarms Revealed by Single Nanoparticle Tracking.

During swarming, high density flagella-driven bacteria migrate collectively in a swirling pattern on wet agar surfaces, immersed in a thin viscous fluid layer called "swarm fluid". Though the fluid environment has essential role in the emergence of swarming behavior, the microscopic mechanisms of it in mediating the cooperation of bacteria populations are not fully understood. Here, instead of micro-sized tracers used in previous research, we use gold nanorods as single particle tracers to probe the dynamics of the swarm fluid. This protocol includes five major parts: (1) the culture of swarming bacterial colony; (2) the preparations of gold nanorod tracers and the micro-spraying technique which are used to put the nanotracers into the upper fluid of bacterial swarms; (3) imaging and tracking; (4) other necessary control experiments; (5) data analysis and fitting of physical models. With this method, the nano-sized tracers could move long distances above motile cells without direct collisions with the bacteria bodies. In this way, the microscopic dynamics of the swarm fluid could be tracked with high spatiotemporal resolution. Moreover, the comprehensive analysis of multi-particle trajectories provides systematic visualization of the fluid dynamics. The method is promising to probe the fluid dynamics of other natural or artificial active matter systems.

Interferometric spectroscopy and high-speed orientation detection of individual gold nanorods.

Although 3D positional tracking of single nanoparticles in suspension is now an established technique, the small size of the particles compared to the wavelength of light has meant it is still challenging to optically characterize individual diffusing particles in other ways. Here we introduce Quantitative Optical Anisotropy Imaging (QOAI), an interferometric technique that fills some of this gap by allowing for real-time tracking of orientation as well as spectroscopic characterization of polarizability in nanoparticles at the microsecond timescale. Applying this to gold nanorods, we demonstrate measurement of nanorod orientation with high precision with simultaneous spectroscopic characterization of the rods' longitudinal plasmon resonance. We also show that we can quantify rotational diffusion in individual particles in both the azimuthal and polar directions near a solid wall, as well as detecting binding of particles to that wall. The simple optical configuration of QOAI will make combining it with positional nanoparticle tracking techniques straightforward, and this opens the door to measurements that are not reachable with current techniques, such as detailed characterization of correlations between rotational and translational diffusion in nanoparticles, real-time observation of particle aggregation and assembly, and measurements of fluctuations in the plasmon resonance in metal nanoparticles as they encounter a changing or heterogeneous environment.

Single Nanoparticle Tracking Reveals Efficient Long-Distance Undercurrent Transport in Upper Fluid of Bacterial Swarms.

SummaryFlagellated bacteria move collectively in a swirling pattern on agar surfaces immersed in a thin layer of viscous “swarm fluid,” but the role of this fluid in mediating the cooperation of the bacterial population is not well understood. Herein, we use gold nanorods (AuNRs) as single particle tracers to explore the spatiotemporal structure of the swarm fluid. Individual AuNRs are moving in a plane of ∼2 μm above swarms, traveling for long distances in high speed without interferences from bacterial movements. The particles are lifted and transported by collective mixing of small vortices around bacteria during localized clustering and de-clustering of motile cells. Their motions fit the Lévy walk model, revealing efficient fluidic flows above the swarms. These flows provide obstacle-free highways for long-range material transportations, allow swarming bacteria to perform population-level communications, and imply the essential role of the fluid phase on the emergence of large-scale synergy.

Supercharging Superlocalization Microscopy: How Electrochemical Charging of Plasmonic Nanostructures Uncovers Hidden Heterogeneity.

Superlocalization microscopy enables the position of single plasmonic nanoparticles to be determined with <25 nm precision, enabling single-nanoparticle tracking and super-resolution imaging experiments to be conducted with sub-diffraction-limited spatial resolution. In many of these applications, the superlocalized position of the nanoparticle is assumed to correspond to the geometric center of the nanoparticle. However, work reported by Wang and co-workers in this issue of ACS Nano suggests that this assumption can be incorrect, based on studies in which electrochemically charging a nanoparticle leads to reproducible shifts in its scattering center. The shift is believed to originate from nonuniform charge accumulation in the nanoparticle, due to the inherent heterogeneity in nanoparticle surface properties. This Perspective explores the implications of this result, both for using this shift to probe dynamic changes in nanoparticle surface chemistry as well as for exploiting nonuniform charge accumulation to promote site-specific chemical reactions.

Identification of Nanoparticles via Plasmonic Scattering Interferometry.

The development of optical imaging techniques has led to significant advancements in single-nanoparticle tracking and analysis, but these techniques are incapable of label-free selective nanoparticle recognition. A label-free plasmonic imaging technology that is able to identify different kinds of nanoparticles in water is now presented. It quantifies the plasmonic interferometric scattering patterns of nanoparticles and establishes relationships among the refractive index, particle size, and pattern both numerically and experimentally. Using this approach, metallic and metallic oxide particles with different radii were distinguished without any calibration. The ability to optically identify and size different kinds of nanoparticles can provide a promising platform for investigating nanoparticles in complex environments to facilitate nanoscience studies, such as single-nanoparticle catalysis and nanoparticle-based drug delivery.

Single-Nanoparticle Tracking with Angstrom Localization Precision and Microsecond Time Resolution

Gold nanoparticles (AuNPs) have been used as a contrast agent for optical imaging of various single biomolecules. Because AuNPs have high scattering efficiency without photobleaching, biomolecular dynamics have been observed with nanometer localization precision and sub-millisecond time resolution. To understand the working principle of biomolecular motors in greater detail, further improvement of the localization precision and time resolution is necessary. Here, we investigated the lower limit of localization precision achievable with AuNPs and the fundamental law, which determines the localization precision. We first used objective-lens-type total internal reflection dark-field microscopy to obtain a scattering signal from an isolated AuNP. The localization precision was inversely proportional to the square root of the photon number, which is consistent with theoretical estimation. The lower limit of precision for a 40 nm AuNP was ∼0.3 nm with 1 ms time resolution and was restricted by detector saturation. To achieve higher localization precision, we designed and constructed an annular illumination total internal reflection dark-field microscopy system with an axicon lens, which can illuminate the AuNPs at high laser intensity without damaging the objective lens. In addition, we used high image magnification to avoid detector saturation. Consequently, we achieved 1.3 Å localization precision for 40 nm AuNPs and 1.9 Å localization precision for 30 nm AuNPs at 1 ms time resolution. Furthermore, even at 33 μs time resolution, localization precisions at 5.4 Å for 40 nm AuNPs and at 1.7 nm for 30 nm AuNPs were achieved. We then observed motion of head of kinesin-1 labeled with AuNP at microsecond time resolution. Transition cycles of bound/unbound states and tethered diffusion of unbound head during stepping motion on microtubule were clearly captured with higher time resolution or smaller AuNP than those used in previous studies, indicating applicability to single-molecule imaging of biomolecular motors.