Quantum Dot Tracking Research Articles

Receptor dimer stabilization by hierarchical plasma membrane microcompartments regulates cytokine signaling.

The interaction dynamics of signaling complexes is emerging as a key determinant that regulates the specificity of cellular responses. We present a combined experimental and computational study that quantifies the consequences of plasma membrane microcompartmentalization for the dynamics of type I interferon receptor complexes. By using long-term dual-color quantum dot (QD) tracking, we found that the lifetime of individual ligand-induced receptor heterodimers depends on the integrity of the membrane skeleton (MSK), which also proved important for efficient downstream signaling. By pair correlation tracking and localization microscopy as well as by fast QD tracking, we identified a secondary confinement within ~300-nm-sized zones. A quantitative spatial stochastic diffusion-reaction model, entirely parameterized on the basis of experimental data, predicts that transient receptor confinement by the MSK meshwork allows for rapid reassociation of dissociated receptor dimers. Moreover, the experimentally observed apparent stabilization of receptor dimers in the plasma membrane was reproduced by simulations of a refined, hierarchical compartment model. Our simulations further revealed that the two-dimensional association rate constant is a key parameter for controlling the extent of MSK-mediated stabilization of protein complexes, thus ensuring the specificity of this effect. Together, experimental evidence and simulations support the hypothesis that passive receptor confinement by MSK-based microcompartmentalization promotes maintenance of signaling complexes in the plasma membrane.

Direct observation of rotation and steps of the archaellum in the swimming halophilic archaeon Halobacterium salinarum

Motile archaea swim using a rotary filament, the archaellum, a surface appendage that resembles bacterial flagella structurally, but is homologous to bacterial type IV pili. Little is known about the mechanism by which archaella produce motility. To gain insights into this mechanism, we characterized archaellar function in the model organism Halobacterium salinarum. Three-dimensional tracking of quantum dots enabled visualization of the left-handed corkscrewing of archaea in detail. An advanced analysis method combined with total internal reflection fluorescence microscopy, termed cross-kymography, was developed and revealed a right-handed helical structure of archaella with a rotation speed of 23 ± 5 Hz. Using these structural and kinetic parameters, we computationally reproduced the swimming and precession motion with a hydrodynamic model and estimated the archaellar motor torque to be 50 pN nm. Finally, in a tethered-cell assay, we observed intermittent pauses during rotation with ∼36° or 60° intervals, which we speculate may be a unitary step consuming a single adenosine triphosphate molecule, which supplies chemical energy of 80 pN nm when hydrolysed. From an estimate of the energy input as ten or six adenosine triphosphates per revolution, the efficiency of the motor is calculated to be ∼6-10%.

3D tracking of single nanoparticles and quantum dots in living cells by out-of-focus imaging with diffraction pattern recognition

Live cells are three-dimensional environments where biological molecules move to find their targets and accomplish their functions. However, up to now, most single molecule investigations have been limited to bi-dimensional studies owing to the complexity of 3d-tracking techniques. Here, we present a novel method for three-dimensional localization of single nano-emitters based on automatic recognition of out-of-focus diffraction patterns. Our technique can be applied to track the movements of single molecules in living cells using a conventional epifluorescence microscope. We first demonstrate three-dimensional localization of fluorescent nanobeads over 4 microns depth with accuracy below 2 nm in vitro. Remarkably, we also establish three-dimensional tracking of Quantum Dots, overcoming their anisotropic emission, by adopting a ligation strategy that allows rotational freedom of the emitter combined with proper pattern recognition. We localize commercially available Quantum Dots in living cells with accuracy better than 7 nm over 2 microns depth. We validate our technique by tracking the three-dimensional movements of single protein-conjugated Quantum Dots in living cell. Moreover, we find that important localization errors can occur in off-focus imaging when improperly calibrated and we give indications to avoid them. Finally, we share a Matlab script that allows readily application of our technique by other laboratories.

Enhanced dimerization drives ligand-independent activity of mutant epidermal growth factor receptor in lung cancer.

Mutations within the epidermal growth factor receptor (EGFR/erbB1/Her1) are often associated with tumorigenesis. In particular, a number of EGFR mutants that demonstrate ligand-independent signaling are common in non-small cell lung cancer (NSCLC), including kinase domain mutations L858R (also called L834R) and exon 19 deletions (e.g., ΔL747-P753insS), which collectively make up nearly 90% of mutations in NSCLC. The molecular mechanisms by which these mutations confer constitutive activity remain unresolved. Using multiple subdiffraction-limit imaging modalities, we reveal the altered receptor structure and interaction kinetics of NSCLC-associated EGFR mutants. We applied two-color single quantum dot tracking to quantify receptor dimerization kinetics on living cells and show that, in contrast to wild-type EGFR, mutants are capable of forming stable, ligand-independent dimers. Two-color superresolution localization microscopy confirmed ligand-independent aggregation of EGFR mutants. Live-cell Förster resonance energy transfer measurements revealed that the L858R kinase mutation alters ectodomain structure such that unliganded mutant EGFR adopts an extended, dimerization-competent conformation. Finally, mutation of the putative dimerization arm confirmed a critical role for ectodomain engagement in ligand-independent signaling. These data support a model in which dysregulated activity of NSCLC-associated kinase mutants is driven by coordinated interactions involving both the kinase and extracellular domains that lead to enhanced dimerization.

Three dimensional time-gated tracking of non-blinking quantum dots in live cells.

Single particle tracking has provided a wealth of information about biophysical processes such as motor protein transport and diffusion in cell membranes. However, motion out of the plane of the microscope or blinking of the fluorescent probe used as a label generally limits observation times to several seconds. Here, we overcome these limitations by using novel non-blinking quantum dots as probes and employing a custom 3D tracking microscope to actively follow motion in three dimensions (3D) in live cells. Signal-to-noise is improved in the cellular milieu through the use of pulsed excitation and time-gated detection.

Quantum dots for quantitative imaging: from single molecules to tissue.

Since their introduction to biological imaging, quantum dots (QDs) have progressed from a little known, but attractive, technology to one that has gained broad application in many areas of biology. The versatile properties of these fluorescent nanoparticles have allowed investigators to conduct biological studies with extended spatiotemporal capabilities that were previously not possible. In this review, we focus on QD applications that provide enhanced quantitative information concerning protein dynamics and localization, including single particle tracking and immunohistochemistry, and finish by examining the prospects of upcoming applications, such as correlative light and electron microscopy and super-resolution. Advances in single molecule imaging, including multi-color and three-dimensional QD tracking, have provided new insights into the mechanisms of cell signaling and protein trafficking. New forms of QD tracking in vivo have allowed the observation of biological processes at molecular level resolution in the physiological context of the whole animal. Further methodological development of multiplexed QD-based immunohistochemistry assays should enable more quantitative analysis of key proteins in tissue samples. These advances highlight the unique quantitative data sets that QDs can provide to further our understanding of biological and disease processes.

Mapping intracellular diffusion distribution using single quantum dot tracking: compartmentalized diffusion defined by endoplasmic reticulum.

The crowded intracellular environment influences the diffusion-mediated cellular processes, such as metabolism, signaling, and transport. The hindered diffusion of macromolecules in heterogeneous cytoplasm has been studied over years, but the detailed diffusion distribution and its origin still remain unclear. Here, we introduce a novel method to map rapidly the diffusion distribution in single cells based on single-particle tracking (SPT) of quantum dots (QDs). The diffusion map reveals the heterogeneous intracellular environment and, more importantly, an unreported compartmentalization of QD diffusions in cytoplasm. Simultaneous observations of QD motion and green fluorescent protein-tagged endoplasmic reticulum (ER) dynamics provide direct evidence that the compartmentalization results from micron-scale domains defined by ER tubules, and ER cisternae form perinuclear areas that restrict QDs to enter. The same phenomenon was observed using fluorescein isothiocyanate-dextrans, further confirming the compartmentalized diffusion. These results shed new light on the diffusive movements of macromolecules in the cell, and the mapping of intracellular diffusion distribution may be used to develop strategies for nanoparticle-based drug deliveries and therapeutics.

Photo Properties and Applications of Single Quantum Dots

As novel fluorescent nanomaterials , quantum dots ( QDs ) have played important roles in many fields , such as chemical analysis , biology sensor , molecular imaging , ow ing to their excellent optical properties. Studies on the properties of single quantum dots can help to find new experimental phenomenology w hich can ' t be found in the ensemble-approach , provide a mentality to improve the properties of quantum dots , contribute to a better application of quantum dots in various fields. In this review , identifications criterion of single quantum dots , photo -properties ( such as fluorescence enhancement , bleaching , blinking , bluing , etc. ) of single quantum dots , and quantum dots application ( such as single quantum dots tracking , single quantum dots biosensor , super- localization technology ) at single nanoparticle levels are commented. The challenges and existing problems of application of single quantum dots are summarized. In the future , synthesis in quantum dots should simultaneously satisfy many excellent photo -properties , including small size , high quantum yield , non-blinking , lager blue shift range , no -biotoxicty . M eanw hile , plasmonic quantum dots that not only exhibit strong fluorescence , but also become excellent probes for surface plasmon scattering , are another significant research field.

Single-molecule optical methods analyzing receptor tyrosine kinase activation in living cells.

Receptor tyrosine kinase activity is typically measured by diverse biochemical methods detecting the amount of phosphorylation of proteins within a cell lysate. In this chapter, we present biophysical methods that allow for studying the activation process of single receptors, in particular the human epidermal growth factor receptor (EGFR) family, in live cells. We describe optical tracking of quantum dot (QD)-labeled single receptors using the total internal reflection fluorescence microscopy (TIRFM), and initial steps of data analysis to identify the time-dependent variation of single-receptor diffusion, which can be widely applied to studying activation of various cell surface receptors.

Delivering stem cells to the healthy heart on biological sutures: effects on regional mechanical function.

Current cardiac cell therapies cannot effectively target and retain cells in a specific area of the heart. Cell-seeded biological sutures were previously developed to overcome this limitation, demonstrating targeted delivery with > 60% cell retention. In this study, both cell-seeded and non-seeded fibrin-based biological sutures were implanted into normal functioning rat hearts to determine the effects on mechanical function and fibrotic response. Human mesenchymal stem cells (hMSCs) were used based on previous work and established cardioprotective effects. Non-seeded or hMSC-seeded sutures were implanted into healthy athymic rat hearts. Before cell seeding, hMSCs were passively loaded with quantum dot nanoparticles. One week after implantation, regional stroke work index and systolic area of contraction (SAC) were evaluated on the epicardial surface above the suture. Cell delivery and retention were confirmed by quantum dot tracking, and the fibrotic tissue area was evaluated. Non-seeded biological sutures decreased SAC near the suture from 0.20 ± 0.01 measured in sham hearts to 0.08 ± 0.02, whereas hMSC-seeded biological sutures dampened the decrease in SAC (0.15 ± 0.02). Non-seeded sutures also displayed a small amount of fibrosis around the sutures (1.0 ± 0.1 mm2 ). Sutures seeded with hMSCs displayed a significant reduction in fibrosis (0.5 ± 0.1 mm2 , p < 0.001), with quantum dot-labelled hMSCs found along the suture track. These results show that the addition of hMSCs attenuates the fibrotic response observed with non-seeded sutures, leading to improved regional mechanics of the implantation region. Copyright © 2014 John Wiley & Sons, Ltd.

Heterogeneous intracellular trafficking dynamics of brain-derived neurotrophic factor complexes in the neuronal soma revealed by single quantum dot tracking.

Accumulating evidence underscores the importance of ligand-receptor dynamics in shaping cellular signaling. In the nervous system, growth factor-activated Trk receptor trafficking serves to convey biochemical signaling that underlies fundamental neural functions. Focus has been placed on axonal trafficking but little is known about growth factor-activated Trk dynamics in the neuronal soma, particularly at the molecular scale, due in large part to technical hurdles in observing individual growth factor-Trk complexes for long periods of time inside live cells. Quantum dots (QDs) are intensely fluorescent nanoparticles that have been used to study the dynamics of ligand-receptor complexes at the plasma membrane but the value of QDs for investigating ligand-receptor intracellular dynamics has not been well exploited. The current study establishes that QD conjugated brain-derived neurotrophic factor (QD-BDNF) binds to TrkB receptors with high specificity, activates TrkB downstream signaling, and allows single QD tracking capability for long recording durations deep within the soma of live neurons. QD-BDNF complexes undergo internalization, recycling, and intracellular trafficking in the neuronal soma. These trafficking events exhibit little time-synchrony and diverse heterogeneity in underlying dynamics that include phases of sustained rapid motor transport without pause as well as immobility of surprisingly long-lasting duration (several minutes). Moreover, the trajectories formed by dynamic individual BDNF complexes show no apparent end destination; BDNF complexes can be found meandering over long distances of several microns throughout the expanse of the neuronal soma in a circuitous fashion. The complex, heterogeneous nature of neuronal soma trafficking dynamics contrasts the reported linear nature of axonal transport data and calls for models that surpass our generally limited notions of nuclear-directed transport in the soma. QD-ligand probes are poised to provide understanding of how the molecular mechanisms underlying intracellular ligand-receptor trafficking shape cell signaling under conditions of both healthy and dysfunctional neurological disease models.

Tracking Receptors Using Individual Fluorescent and Nonfluorescent Nanolabels

The plasma membrane is a fluid-mosaic structure in which some molecules seem to be randomly distributed and others show a precise compartmentalization that is related to their functional properties. These membrane domains are submicrometer in size and therefore are close to or below the optical diffraction limit. This makes their study difficult by conventional microscopy. Moreover, these compartments are usually dynamic in size and composition as their component molecules can continuously enter and exit by diffusion. Real-time, high-resolution, live-imaging methods rather than static imaging are thus required to reflect the real behavior of membrane molecules. Single-molecule techniques fulfill these requirements, as they provide information about the dynamics of molecules, together with nanometer resolution to study their distribution. Here we describe imaging and tracking techniques using nanometer-sized optical labels for the study of the movement of individual or small assemblies of membrane proteins. These labels include fluorescent dyes, luminescent nanocrystals, and absorbing metallic nanoparticles. Single-molecule tracking (SMT), with the use of organic dyes and semiconductor quantum dots (QDs), and single-particle tracking (SPT), with the use of gold nanoparticles, allow one to study the diffusion of individual molecules, their compartmentalization, and their interactions with other molecules. This protocol describes three methods for imaging and tracking membrane proteins: SMT using an organic dye, quantum dot tracking (QDT), and single-nanoparticle photothermal tracking (SNaPT) using gold nanoparticles. Organic dyes and QDs are tracked by single-molecule epifluorescence microscopy. Gold nanoparticles are detected by photothermal heterodyne imaging (PHI) and tracked using a triangulation scheme.

Single Molecule Imaging Reveals that Activating Kinase Domain Mutations Reduce EGFR Mobility and Enhance Dimerization

The epidermal growth factor receptor (EGFR) is a member of the ErbB family of receptor tyrosine kinases which play an important role in a number of physiological responses and have also been identified as a common signaling component in several cancer types. Physiological signaling is initiated by ligand binding which stabilizes EGFR dimers, resulting in auto-phosphorylation of the intracellular kinase domain. Mutation of EGFR is associated with tumor progression due to constitutive activity resulting from several known kinase domain variants. Our previous work has shown that ligand-induced dimerization of wild type EGFR results in decreased receptor mobility, and that this reduction in mobility is dependent on kinase activity. Therefore, we wondered whether ligand-independent activity of EGFR mutants correlates with differences in receptor mobility or dimerization kinetics.Here, we employ multi-color, single particle tracking (SPT) of quantum dots to visualize and quantify the dynamics and interactions of individual receptors within the membrane. Using a novel, high-speed hyperspectral microscope, we are able to track up to eight spectrally distinct quantum dots for visualization and quantification of receptor interactions. From this data, we quantify receptor dimerization kinetics using a Hidden Markov Model. We find that unliganded EGFR mutants exhibit a reduced mobility and longer dimer lifetimes when compared to wild type. These results are consistent with constitutive activity of the EGFR mutants and suggest that the active kinase domain facilitates dimer formation in the absence of ligand. Moreover, we test the effects of clinical tyrosine kinase inhibitors on the single molecule behavior of EGFR mutants. We further examined the mobility and clustering of EGFR within the cellular actin cytoskeleton by hyperspectral tracking over GFP-actin, as well as two-color super-resolution microscopy.

A Bright Light to Reveal Mobility: Single Quantum Dot Tracking Reveals Membrane Dynamics and Cellular Mechanisms.

This perspective describes recent progress in single quantum dot techniques, with an emphasis on their applications in exploring membrane dynamics and cellular mechanisms. In these cases, conventional population measurements, such as fluorescence recovery after photobleaching, yield only a mean value on an ensemble or bulk collection of molecules, where the behavior of individual proteins and vehicles is missing. In recent years, the single quantum dot imaging approach has been introduced as a sub-category of single molecule fluorescent techniques to reveal single protein/vehicle dynamics in real-time. One of the major advantages of using single quantum dots is the high signal-to-noise ratio originating from their unique photophysical properties such as extraordinarily high molar extinction coefficients and large effective Stokes shifts. In addition to a brief overview on the principle of single quantum dot imaging techniques, we highlight recent discoveries and discuss future directions in the field.

Single Quantum Dot Tracking Reveals that an Individual Multivalent HIV-1 Tat Protein Transduction Domain Can Activate Machinery for Lateral Transport and Endocytosis

The mechanisms underlying the cellular entry of the HIV-1 Tat protein transduction domain (TatP) and the molecular information necessary to improve the transduction efficiency of TatP remain unclear due to the technical limitations for direct visualization of TatP's behavior in cells. Using confocal microscopy, total internal reflection fluorescence microscopy, and four-dimensional microscopy, we developed a single-molecule tracking assay for TatP labeled with quantum dots (QDs) to examine the kinetics of TatP initially and immediately before, at the beginning of, and immediately after entry into living cells. We report that even when the number of multivalent TatP (mTatP)-QDs bound to a cell was low, each single mTatP-QD first locally induced the cell's lateral transport machinery to move the mTatP-QD toward the center of the cell body upon cross-linking of heparan sulfate proteoglycans. The centripetal and lateral movements were linked to the integrity and flow of actomyosin and microtubules. Individual mTatP underwent lipid raft-mediated temporal confinement, followed by complete immobilization, which ultimately led to endocytotic internalization. However, bivalent TatP did not sufficiently promote either cell surface movement or internalization. Together, these findings provide clues regarding the mechanisms of TatP cell entry and indicate that increasing the valence of TatP on nanoparticles allows them to behave as cargo delivery nanomachines.

Multi-Color Quantum Dot Tracking Using a High-Speed Hyperspectral Line-Scanning Microscope

Many cellular signaling processes are initiated by dimerization or oligomerization of membrane proteins. However, since the spatial scale of these interactions is below the diffraction limit of the light microscope, the dynamics of these interactions have been difficult to study on living cells. We have developed a novel high-speed hyperspectral microscope (HSM) to perform single particle tracking of up to 8 spectrally distinct species of quantum dots (QDs) at 27 frames per second. The distinct emission spectra of the QDs allows localization with ∼10 nm precision even when the probes are clustered at spatial scales below the diffraction limit. The capabilities of the HSM are demonstrated here by application of multi-color single particle tracking to observe membrane protein behavior, including: 1) dynamic formation and dissociation of Epidermal Growth Factor Receptor dimers; 2) resolving antigen induced aggregation of the high affinity IgE receptor, FcεR1; 3) four color QD tracking while simultaneously visualizing GFP-actin; and 4) high-density tracking for fast diffusion mapping.

Visualization of Plasma Membrane Compartmentalization by High-Speed Quantum Dot Tracking

In this study, we have imaged plasma membrane molecules labeled with quantum dots in live cells using a conventional wide-field microscope with high spatial precision at sampling frequencies of 1.75 kHz. Many of the resulting single molecule trajectories are sufficiently long (up to several thousand steps) to allow for robust single trajectory analysis. This analysis indicates that a majority of the investigated molecules are transiently confined in nanoscopic compartments with a mean size of (100–150 nm)(2) for a mean duration of 50–100 ms.

Three-Dimensional Tracking of Fluorescent Particles in Confocal Microscopy

Three-dimensional tracking of fluorescent particles has the potential of offering scientists much more information pertaining to biological phenomena than can be provided with two-dimensional tracking. In this work, we describe a tracking algorithm that can be used in any optical configuration where a two- or three-dimensional point spread function is sampled in space and time. We show preliminary results from the application of this algorithm to three-dimensional tracking of freely-diffusing fluorescent quantum dots in a confocal microscope. Further, using experimental data, we illustrate how the algorithm can maintain tracking in low-SNR settings where high-background noise may be present or where low-excitation power is required due to concerns of phototoxicity. We describe the versatility of the algorithm in tracking fluorescent particles of various diffusion coefficients and velocities by the adjustment of tuneable parameters. Since the algorithm does not estimate the position of the particle, the user may choose any localization algorithm on the resulting trajectories. In our analysis, we use a GPS-like localization technique called fluoroBancroft to estimate the particle position from the resulting trajectories and describe results from Monte Carlo simulations that indicate optimal window sizes.View Large Image | View Hi-Res Image | Download PowerPoint Slide

Fast 3D Single Molecule Tracking with Multifocal Plane Microscopy in Polarized Epithelia Reveals a Novel Cellular Process of Intercellular Transfer

The study of intracellular trafficking processes represents a fundamental problem in many areas of biomedical research. Single molecule imaging approaches are well suited to study heterogeneous processes in live cells. However, 3D single molecule imaging of intracellular trafficking events in a thick sample such as an epithelial-cell monolayer poses several technical challenges. Specifically, we require a methodology that not only enables fast 3D tracking of single molecules across a cell monolayer, but also enables the imaging of the cellular environment with which the single molecule interacts. Current approaches that are widely used for 3D single molecule imaging/tracking are not well suited for studying the intracellular trafficking pathways due to restricted imaging depth, poor temporal resolution, and the ability to track only a few molecules at one time. Here we show that multifocal plane microscopy (MUM), a 3D imaging modality developed by our group, provides the much needed solution to this longstanding problem by demonstrating fast 3D tracking of quantum dot labeled transferrin molecules in a ∼10 micron thick epithelial cell monolayer.The use of MUM led to the unexpected discovery of a novel cellular process, intercellular transfer, that involves the rapid exchange of Tf molecules between two adjacent cells in the monolayer. We also report 3D single molecule tracking of endocytosis and exocytosis at the lateral plasma membrane of cells in the monolayer. This lateral membrane has been notoriously difficult to image with other cellular imaging modalities. A detailed characterization of these events based on the temporal and 3D intracellular spatial behavior of Tf molecules has been made. The methods and approaches used in this study have broad applicability to investigate 3D trafficking pathways in other cell systems and models.

Electrostatically Controlled Quantum Dot Monofunctionalization for Interrogating the Dynamics of Protein Complexes in Living Cells

Quantum dots (QD) are powerful labels for probing diffusion and interaction dynamics of proteins on the single molecule level in living cells. Protein cross-linking due to multifunctional QD strongly affects these properties. This becomes particularly critical when labeling interaction partners with QDs for interrogating the dynamics of complexes. We have here implemented a generic method for QD monofunctionalization based on electrostatic repulsion of a highly negatively charged peptide carrier. On the basis of this method, monobiotinylated QDs were prepared with high yield as confirmed by single molecule assays. These QDs were successfully employed for probing the assembly and diffusion dynamics of binary and ternary cytokine-receptor complexes on the surface of living cells by dual color single QD tracking. Thus, sequential and dynamic recruitment of the type I interferon receptor subunits by the ligand could be observed.