Single Particle Tracking Techniques Research Articles

Multicolor 3D Single Particle Tracking using Spectrally Displaced Localization

Single particle tracking (SPT) techniques such as sptPALM, uPAINT, and quantum dot tracking have given unprecedented insight into molecular dynamics in living cells. They allow monitoring the behavior of individual proteins in the plasma membrane as well as their molecular interaction with scaffold proteins at millisecond temporal resolution and high spatial resolution (<30 nm) by fitting the point spread function (PSF) of the individual emitter. While these SPT methods have been extended to study the temporal dynamics and co-organization of multiple proteins, conventional experimental setups used to perform multicolor imaging are typically limited to two simultaneous wavelengths. Increasing the number of colors requires additional filters for specific fluorescent tags and is usually performed at the expense of a loss of spatial or temporal resolution. This limits the minimum diffusion coefficient that can be measured, thereby degrading the ability to differentiate between molecular diffusion regimes like immobilization and confined diffusion.By employing a dual collection objective imaging configuration compatible with live cell imaging, we will present a single molecule tracking technique that allows for simultaneous 3D single particle tracking of multiple distinct species without compromising the spatio-temporal resolution. A dispersive element introduced into the second optical path induces a spectrally-dependent displacement, which is used to separate numerous fluorescent species of single emitters based on their emission spectra. We will present the system's spectral separation capabilities in a quantitative manner and validate the technique in fixed samples. We will conclude by demonstrating the ability to simultaneously track several fluorescing species in live cells.

Development of Biophysical Markers That Quantify Metastatic Potentials of Prostate Cancer Cells using Tsunami Microscope

Dysregulated trafficking of receptor tyrosine kinases has been linked to oncogenesis. Here we study the trafficking patterns and dynamics of epidermal growth factor receptor (EGFR) trafficking of benign (BPH1), non-invasive malignant (LNCaP), and highly invasive malignant (PC-3) prostate cells using an advanced 3D single-particle tracking technique termed TSUNAMI (Tracking of Single particles Using Nonlinear And Multiplexed Illumination). As a feedback-control tracking system, TSUNAMI is capable of tracking fluorescent nanoparticle-tagged EGFR for up to 10 minutes and in the z-direction for up to ±50 microns. To analyze the long 3D trajectories generated by the TSUNAMI microscope, a trajectory analysis algorithm is developed to classify trajectories and extract the dynamic parameters, such as diffusivity, inward movement, and internalization duration. These parameters can be used to quantify the metastatic potentials of prostate cancer cell lines. For instance, the diffusivities of EGFRs on the highly invasive malignant PC-3 cells (0.010 ± 0.014 µm2/s) are around one-quarter of those estimated from the benign BPH1 cells (0.036 ± 0.058 µm2/s), possibly due to the abnormally high expression of EGFRs on the PC-3 cells. The highly invasive PC-3 cells also exhibit longer (2.83 ± 0.23 µm vs. 1.45 ± 0.16 µm) and faster (0.021 ± 0.016 µm/s vs. 0.005 ± 0.002 µm/s) inward movement as compared with the non-invasive LNCaP prostate cancer cells, which could be due to the high endocytotic activity associated with the invasive PC-3 cells. In addition, the dynamics parameters extracted from the EGFR trajectories are correlated with the expression levels of a number of epithelial-mesenchymal-transition (EMT)-related genes. The high EGFR expression is related to the decrease of EGFR diffusivity, and the increase of dynamins coheres with more active inward movement. After EMT induction, the non-invasive LNCaP prostate cancer cells also exhibit gene expression profiles and EGFR trafficking patterns similar to those shown in invasive PC-3 cells. Our work demonstrates that EGFR trajectory-derived dynamics parameters are linked to metastatic potentials. A new class of biophysical markers can be established based on the analysis of membrane protein trajectories.

Experimental investigation of slip velocity and settling distribution of micro-particles in converging–diverging microchannel

An experimental test bed based on single particle tracking techniques is employed in order to investigate the velocity domain, slip velocity, and settling distribution of micro-particles in low-Reynolds number poiseuille flow in converging–diverging microchannel. Three-dimensional velocity domain of particles are studied in the presence of walls and compared with the particle-free fluid. The results show that the velocity of particles moving near the side walls of microchannel decreases about 30 % compared to those moving at the centerline. Furthermore, the effects of converging–diverging geometry on sedimentation of micro-particles are considered. The results show an average decrease of about 40 % in sedimentation of particles among the total particles in converging–diverging channels which is one of the main advantages of these channels in comparison with the straight types.

The shape and behaviour of a granular bed in a rotating drum using Eulerian flow fields obtained from PEPT

Non-invasive single-particle tracking techniques, such as positron emission particle tracking (PEPT), provide useful information about the behaviour of a representative particle moving in a bulk of similar particles in a rotating drum. The Lagrangian trajectories that they yield can be used to study, for example, particulate diffusion or granular interaction. However, often the Eulerian flow fields of the entire granular bed are more useful– they can be used to study segregation, for instance, or the evolution of the free surface of the bed. In this work, we present a technique for converting Lagrangian trajectories to Eulerian flow fields via a time-weighted residence time distribution (RTD) of the tracked particle. We then perform PEPT experiments on a mono-disperse bed of spherical particles in a cylindrical drum, rotated at various rates, and use the RTD procedure to obtain flow fields of the bed. We use these flow fields to investigate the effect of drum rotational speed on the shape and behaviour of a granular bed in a rotating drum, and the insights gained thereby to define a comprehensive set of surfaces– such as the bulk free surface– to divide the bed into regions of distinct granular behaviour. We further define scalar bed features– such as the centre of circulation of the bed– that can be used to quantitatively compare the behaviour of granular beds in rotating drums operated under various conditions.

Automatic Five-Dimensional Single Particle Tracking in Live Cells

A novel single particle tracking (SPT) technique has been developed to visualize the intracellular transport of cargos in five dimensions (5D): the three spatial coordinates (x, y, z) and two orientation angles (azimuthal angle and polar angle) of a probe's transition dipole over a series of time steps. Tracking rotational probes, such as gold nanorods, in the z-axis is challenging because translational motions in the z-axis can be easily confused with rotational motions as both types of motion result in similar changes in signal intensity. The 5D-SPT method overcomes this long-standing challenge by utilizing the concept of parallax to sense the axial movement of the target object and employs an automatic feedback loop algorithm to control the objective's focal plane and bring the target object back to focus repetitively. In such an implementation, the target object is kept in focus to provide the highest possible S/N for 2D localization and orientation determination, while the z positions are recorded from the vertical movement of a high-precision objective scanner. Through the live-cell SPT experiments, we demonstrate that the 5D-SPT technique has potential to significantly improve the capacity to study the intracellular transport of cargos in a cellular environment.

Live-Cell Single-Molecule Imaging of Endogenous MRNA in Stress Granules

Eukaryotic mRNA plays a critical role in the production of proteins in cells, transporting the genetic information from the nucleus to the cytoplasm. In this process, mRNA undergoes various regulations. Especially, under environmental stresses such as heat shock or oxidative stress, cytoplasmic mRNAs and some kinds of proteins, including RNA binding proteins and translation initiation factors, assemble into granular structures which are known as stress granules (SGs). Previous studies suggest that SGs work as sites of temporal translation repression, allowing the cells to survive under stress. However, the detailed functions and mechanisms of SGs have been unclear because of the lack of information about the distribution and dynamics of each mRNA at the molecular scale. Here, we utilized single-molecule-sensitive fluorescence microscopy as a tool for investigating the nanoscale spatio-temporal regulations of mRNA in SGs, where fluorescently labeled antisense oligonucleotides enabled us to image endogenous mRNAs. First, we visualized the nanoscale spatial distribution of mRNAs by localization-based super-resolution microscopy that provides us the spatial resolutions of ∼20 nm in xy and ∼60 nm in z, ten times greater than the conventional fluorescence microscopy. As a result, we found that mRNA forms small clusters inside large SGs. Second, we applied single particle tracking technique to investigating the dynamics of endogenous mRNAs. We analyzed the diffusion of mRNAs based on the tracking results and found that mRNA's movement is confined inside SGs, compared to in the cytoplasm outside SGs. Finally, we combined these two methods, super-resolution microscopy and single particle tracking. This combined method revealed that mRNA is anchored to the nanoscale clusters inside SGs, suggesting that these small clusters work as a unit for the translational regulation in SGs.

Improving z-tracking accuracy in the two-photon single-particle tracking microscope.

Here, we present a method that can improve the z-tracking accuracy of the recently invented TSUNAMI (Tracking of Single particles Using Nonlinear And Multiplexed Illumination) microscope. This method utilizes a maximum likelihood estimator (MLE) to determine the particle's 3D position that maximizes the likelihood of the observed time-correlated photon count distribution. Our Monte Carlo simulations show that the MLE-based tracking scheme can improve the z-tracking accuracy of TSUNAMI microscope by 1.7 fold. In addition, MLE is also found to reduce the temporal correlation of the z-tracking error. Taking advantage of the smaller and less temporally correlated z-tracking error, we have precisely recovered the hybridization-melting kinetics of a DNA model system from thousands of short single-particle trajectories in silico. Our method can be generally applied to other 3D single-particle tracking techniques.

Fast Three-Dimensional Single-Particle Tracking in Natural Brain Tissue

Observation of molecular dynamics is often biased by the optical very heterogeneous environment of cells and complex tissue. Here, we have designed an algorithm that facilitates molecular dynamic analyses within brain slices. We adjust fast astigmatism-based three-dimensional single-particle tracking techniques to depth-dependent optical aberrations induced by the refractive index mismatch so that they are applicable to complex samples. In contrast to existing techniques, our online calibration method determines the aberration directly from the acquired two-dimensional image stream by exploiting the inherent particle movement and the redundancy introduced by the astigmatism. The method improves the positioning by reducing the systematic errors introduced by the aberrations, and allows correct derivation of the cellular morphology and molecular diffusion parameters in three dimensions independently of the imaging depth. No additional experimental effort for the user is required. Our method will be useful for many imaging configurations, which allow imaging in deep cellular structures.

Modes of Diffusion of Cholera Toxin Bound to GM1 on Live Cell Membrane by Image Mean Square Displacement Analysis

The image-mean square displacement technique applies the calculation of the mean square displacement commonly used in single-molecule tracking to images without resolving single particles. The image-mean square displacement plot obtained is similar to the mean square displacement plot obtained using the single-particle tracking technique. This plot is then used to reconstruct the protein diffusion law and to identify whether the labeled molecules are undergoing pure isotropic, restricted, corralled, transiently confined, or directed diffusion. In our study total internal reflection fluorescence microscopy images were taken of Cholera toxin subunit B (CtxB) membrane-labeled NIH 3T3 mouse fibroblasts and MDA 231 MB cells. We found a population of CTxB undergoing purely isotropic diffusion and one displaying restricted diffusion with corral sizes ranging from 150 to ∼1800 nm. We show that the diffusion rate of CTxB bound to GM1 is independent of the size of the confinement, suggesting that the mechanism of confinement is different from the mechanism controlling the diffusion rate of CtxB. We highlight the potential effect of continuous illumination on the diffusion mode of CTxB. We also show that aggregation of CTxB/GM1 in large complexes occurs and that these aggregates tend to have slower diffusion rates.

Weak ergodicity breaking and ageing in anomalous diffusion

Modern single particle tracking techniques and many large scale simulations produce time series r(t) of the position of a tracer particle. Standardly these are evaluated in terms of the time averaged mean squared displacement. For ergodic processes such as Brownian motion, one can interpret the results of such an analysis in terms of the known theories for the corresponding ensemble averaged mean squared displacement, if only the measurement time is sufficiently long. In anomalous diffusion processes, that are widely observed over many orders of magnitude, the equivalence between (long) time and ensemble averages may be broken (weak ergodicity breaking). In such cases the time averages may no longer be interpreted in terms of ensemble theories. Here we collect some recent results on weakly non-ergodic systems with respect to the time averaged mean squared displacement and the inherent irreproducibility of individual measurements. We also address the phenomenon of ageing, the dependence of physical observables on the time span between initial preparation of the system and the start of the measurement.

Quantitative Theory of Active Diffusion Trajectories by Instantaneous Diffusion Coefficient

Owing to the development of single-particle tracking techniques, it is able to observe real-time diffusive motions of labelled tiny particles in living cells. To quantify the diffusion trajectories, the mean-squared displacement (MSD) analysis is common and conventional. The diffusion coefficient quantified by the analysis is a good barometer of the mobility. But, in terms of statistical physics, equilibrium and stationarity are assumed in the MSD analysis. Accordingly, the analysis depends on the statistical ensemble. When diffusive motions are driven by non-equilibrium and non-stationary fluctuations as the outcome of biological activity, such fluctuations are averaged and neglected in the MSD analysis. Such enhanced diffusive motion is called active diffusion. To understand the interaction among diffusive motion, biological activity, and environment in detail, we should develop a new diffusion analysis theory beyond the MSD analysis.Hence, our subject is not an ensemble of diffusion trajectories, only individual ones. The MSD analysis is based on the hypothesis of a stationary stochastic process, but our theory is based on stochastic energetics, which is thermodynamics in fluctuating small world.Here, I will show energetics of single active diffusion trajectories, where the instantaneous diffusion coefficient (IDC) as a new diffusion analysis quantity is derived. Considering decomposition of energy dissipation in a non-equilibrium steady state, we can make implication of the IDC clear. An advantage of our theory is to be able to discuss the meso scale energetics from only single-particle tracking data without measuring response.

Trajectory-based co-localization measures for nanoparticle-cell interaction studies.

High-resolution live cell microscopy will soon have a fundamental role in understanding bio-nano interactions, providing material that can be exploited using single particle tracking techniques. The present work uses 3D timelapse images obtained with confocal microscopy, to temporally resolve the co-localization between polystyrene nanoparticles and lysosomes in live cells through object-based measurements.

Three-Dimensional Mapping of mRNA Export through the Nuclear Pore Complex

The locations of transcription and translation of mRNA in eukaryotic cells are spatially separated by the nuclear envelope (NE). Plenty of nuclear pore complexes (NPCs) embedded in the NE function as the major gateway for the export of transcribed mRNAs from the nucleus to the cytoplasm. Whereas the NPC, perhaps one of the largest protein complexes, provides a relatively large channel for macromolecules to selectively pass through it in inherently three-dimensional (3D) movements, this channel is nonetheless below the diffraction limit of conventional light microscopy. A full understanding of the mRNA export mechanism urgently requires real-time mapping of the 3D dynamics of mRNA in the NPC of live cells with innovative imaging techniques breaking the diffraction limit of conventional light microscopy. Recently, super-resolution fluorescence microscopy and single-particle tracking (SPT) techniques have been applied to the study of nuclear export of mRNA in live cells. In this review, we emphasize the necessity of 3D mapping techniques in the study of mRNA export, briefly summarize the feasibility of current 3D imaging approaches, and highlight the new features of mRNA nuclear export elucidated with a newly developed 3D imaging approach combining SPT-based super-resolution imaging and 2D-to-3D deconvolution algorithms.

Super-resolved and dynamic imaging of membrane proteins in plant cells reveal contrasting kinetic profiles and multiple confinement mechanisms.

Dear Editor, Microscopic techniques allow either a global mobility analysis of proteins with fluorescence recovery after photobleaching (FRAP) or single-protein mobility characterization with single-particle or quantum dot tracking.For instance,total internal reflection fluorescence microscopy allowed single-particle tracking (SPT) of Arabidopsis plasma membrane (PM) proteins,revealing their heterogeneous distribution,low lateral diffusion,and dynamic properties in response to salt stress (Li et al.,2011).Studies of SPT based on green fluorescent protein are unfortunately restricted by the density of proteins at the surface,since diffracted emission fluorescence prevents tracking of individual proteins separated by less than 1 μm.The recent emergence of high-density SPT techniques based on temporal emission decorrelation,such as single-particle tracking with photoactivated localization microscopy (sptPALM),allowed the diffraction limit of classic light microscopy to be broken and reach nanometerlevel spatial resolutions (Manley et al.,2008).Application of these techniques has rendered possible the characterization of the structural and dynamic heterogeneity of PM proteins with an accuracy of ～20-80 nm (Rossier et al.,2012).Such super-resolved dynamic imaging of membrane proteins has not yet been applied to any plant system.Here,we report the first use in plants of the live-cell sptPALM technique,providing a high-density super-resolved nanoscale map of individual membrane-protein motions.

Three-dimensional tracking of Rab5- and Rab7-associated infection process of influenza virus.

Three-dimensional (3D) single-particle tracking (SPT) techniques have been widely reported. However, the 3D SPT technique remains poorly used for solving actual biological problems. In this work, a quantum dots (QDs)-based single-particle tracking technique is utilized to explore the Rab5- and Rab7-associated infection behaviors of influenza virus in three dimensions with a set of easily-attained equipment by the fast and accurate centroid method for 3D SPT. The experimental results indicate that Rab5 protein takes part in the virus infection process from the cell periphery to the perinuclear region, while Rab7 protein is mainly involved in the intermittent and confined movements of the virus in the perinuclear region. Evidently, the transition process of the virus-containing vesicles from early to late endosomes might occur during the intermittent movement in the perinuclear region. These findings reveal distinct dynamic behaviors of Rab5- and Rab7-positive endosomes in the course of the intracellular transport of viruses. This work is helpful in understanding the intracellular transport of cargoes.

Globally visualizing the microtubule-dependent transport behaviors of influenza virus in live cells.

Understanding the microtubule-dependent behaviors of viruses in live cells is very meaningful for revealing the mechanisms of virus infection and endocytosis. Herein, we used a quantum dots-based single-particle tracking technique to dynamically and globally visualize the microtubule-dependent transport behaviors of influenza virus in live cells. We found that the intersection configuration of microtubules can interfere with the transport behaviors of the virus in live cells, which lead to the changing and long-time pausing of the transport behavior of viruses. Our results revealed that most of the viruses moved along straight microtubules rapidly and unidirectionally from the cell periphery to the microtubule organizing center (MTOC) near the bottom of the cell, and the viruses were confined in the grid of microtubules near the top of the cell and at the MTOC near the bottom of the cell. These results provided deep insights into the influence of entire microtubule geometry on the virus infection.

Exploring Sialic Acid Receptors‐Related Infection Behavior of Avian Influenza Virus in Human Bronchial Epithelial Cells by Single‐Particle Tracking

Human respiratory tract epithelial cells are the portals of human infection with influenza viruses. However, the infection pathway of individual avian influenza viruses in human respiratory cells remains poorly reported so far. The single-particle tracking technique (SPT) is a powerful tool for studying the transport mechanism of biomolecules in live cells. In this work, we use quantum dots to label avian influenza H9N2 virus and elaborate on the infection mechanism of the virus in human bronchial epithelial (HBE) cells using a three-dimensional SPT technique. We have found that the H9N2 virus can infect HBE cells directly and the virus infection follows an actin filament- and microtubule-dependent process with a three-stage pattern. The transport behaviors show a high degree of consistency between the sialic acid receptors and the influenza virus. Real-time SPT provides dynamic evidence of the sialic acid receptors-related infection behavior of the avian influenza virus in live cells. The study of the influence of sialic acid receptors on virus infection may contribute to a better understanding of the cross-species transmission of the avian influenza virus.

Troika of single particle tracking programing: SNR enhancement, particle identification, and mapping.

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.

High Density Single Particle Tracking with Various Probes

Single Particle Tracking (SPT) is a set of single molecule techniques that identify and characterize the motion of individual particles in a medium. High Density Single Particle Tracking (HDSPT) is a subset of advanced SPT techniques designed to overcome some of the limitations of current SPT methods, primarily to compensate for poor statistical information due to low trajectory counts per observed area as seen in conventional SPT. Quantum Dots (QDs), Fluorogen Activating Peptides (FAPs), and Organic Dyes are recognized probes that have been used in SPT experiments. QDs are very bright, photo stable probes with blinking dictated by power law behaviors. A FAP will bind to a receptor and activate, emitting photons at a constant rate and intensity until it bleaches. ODs have emission behaviors characterized by Jablonski diagrams; depending on the particular OD and buffer combination, they may blink or bleach. Variable emission dynamics provide a challenge in SPT analysis because it requires localization techniques that can properly account for varying probe behaviors. We have modified our multi-emitter fitting algorithm [1] with a Bayesian approach in order to localize overlapping probes with variable intensity profiles. This advancement in localization capturing allows us to overcome one of the more difficult challenges present in high density SPT- reliable probe identification in high density regions. We have tested our modified SPT software on simulated data as well as on live Rat Basophil Leukemia (RBL) cells with various probes to demonstrate our approach to high density SPT.[1] Fang Huang, Samantha L. Schwartz, Jason M. Byars, and Keith A. Lidke, Biomedical Optics Express, Vol. 2, Issue 5, pp. 1377-1393 (2011).

Ion-Specific Effects Modulate the Diffusive Mobility of Colloids in an Extracellular Matrix Gel

The diffusion of colloids in complex biological hydrogels is regulated by a broad range of factors including geometric constraints and different types of physical interactions between the particles and the hydrogel constituents. As a consequence, the particle mobility depends not only on the hydrogel microarchitecture but also on the detailed chemical composition of the hydrogel solvent. Here, we employ single particle tracking techniques to quantify the diffusion behavior of submicrometer-sized particles in such a biological hydrogel. We observe three states of colloid mobility: free diffusion, tightly and weakly bound particles, and transitions between those states. Finally, by comparing the efficiency of particle trapping in Matrigel as a function of the ionic strength of the hydrogel buffer, we show that ion-specific effects regulate the efficiency of this trapping process.