Tracking Of Gold Nanoparticles Research Articles

Single‐cell/nanoparticle trajectories reveal two‐tier Lévy‐like interactions across bacterial swarms

AbstractSwarming bacteria can organize themselves into different local communities, ranging from active cell rafts to reticular biofilms. How millions of motile and immotile cells are dynamically linked together and still function as a whole is a critical interdisciplinary issue. Besides biochemical and molecular characterizations, concerns on the microscopic communication mechanisms underlying the complicated microsystem have been largely simplified to biophysics or physical interactions between individual isolated bacteria. Herein, by single‐cell tracking of fluorescent bacteria in the motile cell layer and single‐particle tracking of gold nanoparticles (AuNPs) in the upper fluid layer in different regions across a swarming colony of Bacillus subtilis, we studied collective multibacteria interactions, using the upper fluid as the medium, in detail. While the dynamic properties differ spatially, both cell migrations and AuNP transports in the swarming edge, the intermediate and the biofilm‐like center regions of the colony can be described with the Lévy‐walk‐like superdiffusion model. Moreover, the speed and motion range of the AuNPs are always much smaller than the bacteria, indicating that the intercellular fluid flow alone cannot explain long‐range cell‐to‐cell signaling. Referring to previous literature, we propose the presence of a decentralized two‐tier “active‐mixing” network in the bacterial community for efficient information exchange.

Effects of Gold Nanoparticles on the Stepping Trajectories of Kinesin.

A substantial increase in the temporal resolution of the stepping of dimeric molecular motors is possible by tracking the position of a large gold nanoparticle (GNP) attached to a labeled site on one of the heads. This technique was employed to measure the stepping trajectories of conventional kinesin (Kin1) using the time-dependent position of the GNP as a proxy. The trajectories revealed that the detached head always passes to the right of the head that is tightly bound to the microtubule (MT) during a step. In interpreting the results of such experiments, it is assumed that the GNP does not significantly alter the diffusive motion of the detached head. We used coarse-grained simulations of a system consisting of the MT-Kin1 complex with and without attached GNP to investigate how the stepping trajectories are affected. The two significant findings are: (1) The GNP does not faithfully track the position of the stepping head, and (2) the rightward bias is typically exaggerated by the GNP. Both these findings depend on the precise residue position to which the GNP is attached. Surprisingly, the stepping trajectories of kinesin are not significantly affected if, in addition to the GNP, a 1 μm diameter cargo is attached to the coiled coil. Our simulations suggest the effects of the large probe have to be considered when inferring the stepping mechanisms using GNP tracking experiments.

Label-Free Visualization and Tracking of Gold Nanoparticles in Vasculature Using Multiphoton Luminescence.

Gold nanoparticles continue to generate interest for use in several biomedical applications. Recently, researchers have been focusing on exploiting their dual diagnostic/therapeutic theranostic capabilities. Before clinical translation can occur, regulatory agencies will require a greater understanding of their biodistribution and safety profiles post administration. Previously, the real-time identification and tracking of gold nanoparticles in free-flowing vasculature had not been possible without extrinsic labels such as fluorophores. Here, we present a label-free imaging approach to examine gold nanoparticle (AuNP) activity within the vasculature by utilizing multiphoton intravital microscopy. This method employs a commercially available multiphoton microscopy system to visualize the intrinsic luminescent signal produced by a multiphoton absorption-induced luminescence effect observed in single gold nanoparticles at frame rates necessary for capturing real-time blood flow. This is the first demonstration of visualizing unlabeled gold nanoparticles in an unperturbed vascular environment with frame rates fast enough to achieve particle tracking. Nanoparticle blood concentration curves were also evaluated by the tracking of gold nanoparticle flow in vasculature and verified against known pre-injection concentrations. Half-lives of these gold nanoparticle injections ranged between 67 and 140 s. This label-free imaging approach could provide important structural and functional information in real time to aid in the development and effective analysis of new metallic nanoparticles for various clinical applications in an unperturbed environment, while providing further insight into their complex uptake and clearance pathways.

Insights into Kinesin-1 Stepping from Simulations and Tracking of Gold Nanoparticle-Labeled Motors

High-resolution tracking of gold nanoparticle-labeled proteins has emerged as a powerful technique for measuring the structural kinetics of processive enzymes and other biomacromolecules. These techniques use point spread function (PSF) fitting methods borrowed from single-molecule fluorescence imaging to determine molecular positions below the diffraction limit. However, compared to fluorescence, gold nanoparticle tracking experiments are performed at significantly higher frame rates and utilize much larger probes. In the current work, we use Brownian dynamics simulations of nanoparticle-labeled proteins to investigate the regimes in which the fundamental assumptions of PSF fitting hold and where they begin to break down. We find that because gold nanoparticles undergo tethered diffusion around their anchor point, PSF fitting cannot be extended to arbitrarily fast frame rates. Instead, camera exposure times that allow the nanoparticle to fully populate its stationary positional distribution achieve a spatial averaging that increases fitting precision. We furthermore find that changes in the rotational freedom of the tagged protein can lead to artifactual translations in the fitted particle position. Finally, we apply these lessons to dissect a standing controversy in the kinesin field over the structure of a dimer in the ATP waiting state. Combining new experiments with simulations, we determine that the rear kinesin head in the ATP waiting state is unbound but not displaced from its previous microtubule binding site and that apparent differences in separately published reports were simply due to differences in the gold nanoparticle attachment position. Our results highlight the importance of gold conjugation decisions and imaging parameters to high-resolution tracking results and will serve as a useful guide for the design of future gold nanoparticle tracking experiments.

Insights into Kinesin-1 Stepping Dynamics from Brownian Dynamics Simulations and High-Resolution Tracking of Gold Nanoparticle-Labeled Motors

Abstract High-resolution tracking of gold nanoparticle-labeled proteins has emerged as a powerful technique for measuring the structural kinetics of processive enzymes and other biomacromolecules. These techniques use point-spread-function fitting methods borrowed from single-molecule fluorescence imaging to determine molecular positions below the diffraction limit. However, compared to fluorescence, gold nanoparticle tracking experiments are performed at significantly higher frame rates and utilize much larger probes. In the current work, we use Brownian dynamics simulations of nanoparticle-labeled proteins to investigate the regimes in which the fundamental assumptions of point-spread-function fitting hold and where they begin to break down. We find that because gold nanoparticles undergo tethered diffusion around their anchor point, point spread function fitting cannot be extended to arbitrarily fast frame rates. Instead, camera exposure times that allow the nanoparticle to fully populate its stationary positional distribution achieve a spatial averaging that increases fitting precision. We furthermore find that changes in the rotational freedom of the tagged protein can lead to artifactual translations in fitted particle position. Finally, we apply these lessons to dissect a standing controversy in the kinesin field over the structure of a dimer in the ATP waiting state. Combining new experiments with simulations, we determine that the rear kinesin head in the ATP waiting state is unbound but not displaced from its previous microtubule binding site, and that apparent differences in separately published reports were simply due to differences in the gold nanoparticle attachment position. Our results highlight the importance of gold conjugation decisions and imaging parameters to high-resolution tracking results, and will serve as a useful guide for the design of future gold nanoparticle tracking experiments.

Intracellular localization of gold nanoparticles with targeted delivery in MT-4 lymphocytes

The clinical utility of important therapeutic agents is often limited by the poor permeability of biological membranes. Cell penetrating peptides are usually employed to circumvent this challenge. This approach, coupled with gold nanoparticles, are a promising vehicle for drug delivery due to its good biocompatibility profile, negligable toxicity and possibility for multi-functionalization. Here we report the functionalization and intracellular tracking of gold nanoparticles decorated with a TAT cell penetrating peptide and a fluorescein tag in a simple, two step process. Fluorescence microscopy has confirmed the localization of the functionalized nanoparticles to be inside the cells, specifically within, or in close proximity to the nuclei of MT-4 lymphocytes; a HIV-relevant cell line in which this has not been previously demonstrated. The results of this study demonstrate that TAT has been efficiently conjugated to gold nanoparticles to facilitate both cellular and targeted nuclear entry.

Visualization of lipids and proteins at high spatial and temporal resolution via interferometric scattering (iSCAT) microscopy

Microscopy based on the interferometric detection of light scattered from nanoparticles (iSCAT) was introduced in our laboratory more than a decade ago. In this work, we present various capabilities of iSCAT for biological studies by discussing a selection of our recent results. In particular, we show tracking of lipid molecules in supported lipid bilayers (SLBs), tracking of gold nanoparticles with diameters as small as 5 nm and at frame rates close to 1 MHz, 3D tracking of Tat peptide-coated nanoparticles on giant unilamellar vesicles (GUVs), imaging the formation of lipid bilayers, sensing single unlabelled proteins and tracking their motion under electric fields, as well as challenges of studying live cell membranes. These studies set the ground for future quantitative research on dynamic biophysical processes at the nanometer scale.

Iodine-125-labeled cRGD-gold nanoparticles as tumor-targeted radiosensitizer and imaging agent.

Research interests on radiosensitive property of gold nanoparticles (GNPs) are rapidly raised because of the extensively proved in vitro effectiveness and clinical necessity. However, the issue of targeted accumulation of GNPs in tumor tissues hindered the transference to in vivo applications. In this study, hybrid nano-sized cyclic Arg-Gly-Asp-conjugated GNPs (cRGD-GNPs) integrated with radioactive iodine-125 was fabricated as tumor-targeted radiosensitizer. Therapeutic effects, including acute apoptosis (2 days post treatment) and long-term influence (up to 21 days), were investigated on NCI-H446 tumor-bearing mice via Tc-99 m-Annexin V SPECT and volume measurements, respectively. Apoptosis and volume loss were consistent in showing that tumor growth was effectively suppressed via the treatment of 125I-cRGD-GNP sensitized radiotherapy (RT), a more significantly radiosensitive effect than the treatment of non-targeted GNPs with RT, RT treatment alone, and no treatment. SPECT/CT images showed that the uptake of cRGD-GNPs by tumor tissues reached the peak target/non-target value of 4.76 at around 2 h post injection, and dynamic radioactivity monitoring showed that 125I-cRGD-GNPs maintained about 2.5% of injected dosage at 55 h post injection. For long-term influence, a significant radiosensitized RT-induced volume loss was observed. Hence, cyclic RGD conjugation makes the GNP-based radiosensitizer tumor targeting, offering a new modality for enhancing radiotherapeutic efficacy. Additionally, the introduction of I-125 serves as both a therapeutic factor and a radiotracer for in vivo tracking of GNPs.

Exploring the Chromatin Architecture in Living Cells by Minutes-Long Tracking of Gold Nanoparticles

Investigating the chromatin compaction on the micro(nano)-meter scale, has become a question of interest to understand many cellular processes. Previous evidence suggests that the cell nucleus is spatially heterogeneous and with inaccessible regions mainly due to a high concentration of chromatin. However, the in vivo 3D picture of the nuclear structure remains unclear. In this work, we studied chromatin organization applying the orbital 3D tracking technique to 20 nm gold nanoparticles (NPs) previously incorporated inside the nucleus of NIH3T3 live cells. We have recently shown that metallic NPs do not bleach or blink upon continuous illumination, are extremely stable, very bright and their luminescence spans over the visible spectrum. These characteristics allow us to track them for minutes thus providing 3D trajectories appreciably longer than those based on fluorescent proteins or quantum dots. For this study we have analyzed the motion of 60 NPs. Each one provided us with a ∼5 - 30 minutes long trajectory. In ∼30% of the cases, we have observed that the NPs remain in regions of apparent confined motion (clusters) and eventually they undergo a long (in the micrometer range) excursion. We have found that the NPs always move faster within the clusters but slower while travelling between two clusters. These results suggest that the NPs get trapped into cavities where they can move relatively fast and eventually get transported (as seen by MSD analysis) from one cavity to the next one along segments of slower diffusion. Additionally, in all the cases analyzed, the NPs showed an increased intensity while moving between two cavities.This work was supported by the National Center for Research Resources (NCRR, 5P41RR003155) the National Institute of General Medical Sciences (NIGMS, 8P41GM103540) and the University of California, Irvine.

Single-Molecule Gold-Nanoparticle Tracking

Gold nanoparticles, like single fluorophores, can be used to locate single molecules with nanometer accuracy. Unlike an optical trap, the gold particle label does not exert an external load, which is important for studying diffusive processes. Thus, a gold particle can be used analogously to a single fluorophore, providing similar information but with submillisecond time resolution. The features of gold-nanoparticle tracking (high temporal resolution, small label size, and lack of applied force) facilitate the characterization of structural properties of short-lived intermediates, as shown by our work with myosin V. This protocol provides details for gold-nanoparticle-tracking experiments, including flow cell construction, microscopy, and data analysis, along with a brief outline of actin and myosin preparation. Although details particular to our experiment are given, the approach should be generally applicable.

Nanometer-scale optical imaging of collagen fibers using gold nanoparticles.

We describe 3D single particle tracking of gold nanoparticles (AuNPs) moving along collagen fibers in aqueous environment with two-photon excitation conditions. The photoacoustic effect at the collagen fiber caused by the irradiation with ultrashort, near-infrared laser pulses propels the particles adsorbed to the surface of the collagen fibers. We report the tracking of individual AuNPs in three dimensions with high spatial and temporal resolution, of few nanometers and milliseconds, respectively. Due to the emission signal caused by the interaction between the AuNPs and the weak chromophores in the collagen fiber, the trajectories of individual AuNPs reveal the fiber topography with nanometric resolution. The intensity along the trajectory shows that we are sensitive to the distribution of the weak chromophores on the fiber.

Contribution of the myosin VI tail domain to processive stepping and intramolecular tension sensing

Myosin VI is proposed to act as both a molecular transporter and as an anchor in vivo. A portion of the molecule C-terminal to the canonical lever arm, termed the medial tail (MT), has been proposed to act as either a lever arm extension or as a dimerization motif. We describe constructs in which the MT is interrupted by a glycine-rich molecular swivel. Disruption of the MT results in decreased processive run lengths measured using single-molecule fluorescence microscopy and a decreased step size under applied load as measured in an optical trap. We used single-molecule gold nanoparticle tracking and optical trapping to examine the mechanism of coordination between the heads of dimeric myosin VI. We detect two rate-limiting kinetic processes at low (< 200 micromolar) ATP concentrations. Our data can be explained by a model in which intramolecular tension greatly increases the affinity of the lead head for ADP, likely by slowing ADP release from the lead head. This mechanism likely increases both the motor's processivity and its ability to act as an anchor under physiological conditions.

Contribution of the Myosin VI Tail Domain to Processive Stepping and Intramolecular Tension Sensing

Myosin VI is proposed to act as both a molecular transporter and as a cytoskeletal anchor in vivo. The structural traits and kinetic mechanisms by which myosin VI takes processive, ∼36 nm steps along actin are controversial. In particular, the portion of the molecule C-terminal to the canonical lever arm, termed the medial tail (MT), has been hypothesized to act as either a lever arm extension or as a dimerization motif. We created constructs in which the MT is interrupted by glycine-rich molecular swivels in order to test competing models of the MT's contribution to processive stepping. Disruption of the MT results in decreased processive run lengths measured using single-molecule fluorescence microscopy and a decreased step size under applied load as measured in an optical trap (see Figure). We used single-molecule gold nanoparticle tracking and optical trapping to examine the mechanism of coordination between the heads of dimeric myosin VI. We conclude that intramolecular tension prevents ADP release from the lead head. This mechanism likely increases both the motor's processivity and its ability to act as an anchor under physiological conditions.View Large Image | View Hi-Res Image | Download PowerPoint Slide

Structural and Mechanistic Determinants of Myosin VI Processivity and Anchoring

Myosin VI is a molecular motor that can function both as a transporter and anchor in cells. Its role is regulated through load, being capable of taking multiple consecutive 36 nm steps along an actin filament under zero load and switching to an anchor by stalling when placed under piconewton levels of load. The parameters necessary for such processivity and anchoring are not fully understood. We use high-speed gold nanoparticle tracking to study single molecules of myosin VI with millisecond resolution in the absence of load. Optical tweezers are used to observe the behavior of the molecules when perturbed by load. In order to probe the contribution of the myosin VI tail domain to processivity and anchoring, we have created and characterized a number of mutant tail domain constructs. Our results reveal the resiliency of myosin VI as a transporter and suggest that it has evolved its unusual tail domain for purposes other than efficient cargo transport in the absence of applied load. We present preliminary data investigating the role of the myosin VI tail domain in important cellular processes such as transport against load and load-induced anchoring.

Organelle Tracking in a Living Cell with Microsecond Time Resolution and Nanometer Spatial Precision

The study of cellular processes such as organelle transport often demands particle tracking with microsecond time-resolution and nanometer spatial precision, posing significant challenges to existing tracking methods. Here, we have developed a novel strategy for two-dimensional tracking of gold nanoparticles (GNPs) with 25 mus time resolution and approximately 1.5 nm spatial precision, by using a quadrant photodiode to record the positions of GNPs in an objective-type dark-field microscope. In combination with a feedback loop, this technique records long, high time-resolution and spatial precision trajectories of endocytosed GNPs transported by the molecular motors kinesin and dynein in a living cell. In the full range of organelle velocities (0-8 microm s(-1)), we clearly resolve the individual 8 nm steps of cargoes carried by kinesin, and the 8, 12, 16, 20, and 24 nm steps of those carried by dynein. These experiments yield new information about molecular motor stepping in living cells.