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1
Hou, Shangguo, Courtney Johnson, and Kevin Welsher. "Real-Time 3D Single Particle Tracking: Towards Active Feedback Single Molecule Spectroscopy in Live Cells." Molecules 24, no. 15 (August 2, 2019): 2826. http://dx.doi.org/10.3390/molecules24152826.
Анотація:
Single molecule fluorescence spectroscopy has been largely implemented using methods which require tethering of molecules to a substrate in order to make high temporal resolution measurements. However, the act of tethering a molecule requires that the molecule be removed from its environment. This is especially perturbative when measuring biomolecules such as enzymes, which may rely on the non-equilibrium and crowded cellular environment for normal function. A method which may be able to un-tether single molecule fluorescence spectroscopy is real-time 3D single particle tracking (RT-3D-SPT). RT-3D-SPT uses active feedback to effectively lock-on to freely diffusing particles so they can be measured continuously with up to photon-limited temporal resolution over large axial ranges. This review gives an overview of the various active feedback 3D single particle tracking methods, highlighting specialized detection and excitation schemes which enable high-speed real-time tracking. Furthermore, the combination of these active feedback methods with simultaneous live-cell imaging is discussed. Finally, the successes in real-time 3D single molecule tracking (RT-3D-SMT) thus far and the roadmap going forward for this promising family of techniques are discussed.
2
Speckner, Konstantin, and Matthias Weiss. "Single-Particle Tracking Reveals Anti-Persistent Subdiffusion in Cell Extracts." Entropy 23, no. 7 (July 13, 2021): 892. http://dx.doi.org/10.3390/e23070892.
Анотація:
Single-particle tracking (SPT) has become a powerful tool to quantify transport phenomena in complex media with unprecedented detail. Based on the reconstruction of individual trajectories, a wealth of informative measures become available for each particle, allowing for a detailed comparison with theoretical predictions. While SPT has been used frequently to explore diffusive transport in artificial fluids and inside living cells, intermediate systems, i.e., biochemically active cell extracts, have been studied only sparsely. Extracts derived from the eggs of the clawfrog Xenopus laevis, for example, are known for their ability to support and mimic vital processes of cells, emphasizing the need to explore also the transport phenomena of nano-sized particles in such extracts. Here, we have performed extensive SPT on beads with 20 nm radius in native and chemically treated Xenopus extracts. By analyzing a variety of distinct measures, we show that these beads feature an anti-persistent subdiffusion that is consistent with fractional Brownian motion. Chemical treatments did not grossly alter this finding, suggesting that the high degree of macromolecular crowding in Xenopus extracts equips the fluid with a viscoelastic modulus, hence enforcing particles to perform random walks with a significant anti-persistent memory kernel.
3
Travers, Théo, Vincent G. Colin, Matthieu Loumaigne, Régis Barillé, and Denis Gindre. "Single-Particle Tracking with Scanning Non-Linear Microscopy." Nanomaterials 10, no. 8 (August 3, 2020): 1519. http://dx.doi.org/10.3390/nano10081519.
Анотація:
This study describes the adaptation of non-linear microscopy for single-particle tracking (SPT), a method commonly used in biology with single-photon fluorescence. Imaging moving objects with non-linear microscopy raises difficulties due to the scanning process of the acquisitions. The interest of the study is based on the balance between all the experimental parameters (objective, resolution, frame rate) which need to be optimized to record long trajectories with the best accuracy and frame rate. To evaluate the performance of the setup for SPT, several basic estimation methods are used and adapted to the new detection process. The covariance-based estimator (CVE) seems to be the best way to evaluate the diffusion coefficient from trajectories using the specific factors of motion blur and localization error.
4
Rose, Katie A., Daeyeon Lee, and Russell J. Composto. "pH-Mediated nanoparticle dynamics in hydrogel nanocomposites." Soft Matter 17, no. 10 (2021): 2765–74. http://dx.doi.org/10.1039/d0sm02213f.
Анотація:
The effect of static silica particles on the dynamics of quantum dot (QD) nanoparticles grafted with a poly(ethylene glycol) (PEG) brush in hydrogel nanocomposites is investigated using single particle tracking (SPT).
5
Zhong, Yaning, and Gufeng Wang. "Three-Dimensional Single Particle Tracking and Its Applications in Confined Environments." Annual Review of Analytical Chemistry 13, no. 1 (June 12, 2020): 381–403. http://dx.doi.org/10.1146/annurev-anchem-091819-100409.
Анотація:
Single particle tracking (SPT) has proven to be a powerful technique in studying molecular dynamics in complicated systems. We review its recent development, including three-dimensional (3D) SPT and its applications in probing nanostructures and molecule-surface interactions that are important to analytical chemical processes. Several frequently used 3D SPT techniques are introduced. Especially of interest are those based on point spread function engineering, which are simple in instrumentation and can be easily adapted and used in analytical labs. Corresponding data analysis methods are briefly discussed. We present several important case studies, with a focus on probing mass transport and molecule-surface interactions in confined environments. The presented studies demonstrate the great potential of 3D SPT for understanding fundamental phenomena in confined space, which will enable us to predict basic principles involved in chemical recognition, separation, and analysis, and to optimize mass transport and responses by structural design and optimization.
6
Clarke, David T., and Marisa L. Martin-Fernandez. "A Brief History of Single-Particle Tracking of the Epidermal Growth Factor Receptor." Methods and Protocols 2, no. 1 (January 30, 2019): 12. http://dx.doi.org/10.3390/mps2010012.
Анотація:
Single-particle tracking (SPT) has been used and developed over the last 25 years as a method to investigate molecular dynamics, structure, interactions, and function in the cellular context. SPT is able to show how fast and how far individual molecules move, identify different dynamic populations, measure the duration and strength of intermolecular interactions, and map out structures on the nanoscale in cells. In combination with other techniques such as macromolecular crystallography and molecular dynamics simulation, it allows us to build models of complex structures, and develop and test hypotheses of how these complexes perform their biological roles in health as well as in disease states. Here, we use the example of the epidermal growth factor receptor (EGFR), which has been studied extensively by SPT, demonstrating how the method has been used to increase our understanding of the receptor’s organization and function, including its interaction with the plasma membrane, its activation, clustering, and oligomerization, and the role of other receptors and endocytosis. The examples shown demonstrate how SPT might be employed in the investigation of other biomolecules and systems.
7
Reina, Francesco, John M. A. Wigg, Mariia Dmitrieva, Joël Lefebvre, Jens Rittscher, and Christian Eggeling. "TRAIT2D: a Software for Quantitative Analysis of Single Particle Diffusion Data." F1000Research 10 (August 20, 2021): 838. http://dx.doi.org/10.12688/f1000research.54788.1.
Анотація:
Single particle tracking (SPT) is one of the most widely used tools in optical microscopy to evaluate particle mobility in a variety of situations, including cellular and model membrane dynamics. Recent technological developments, such as Interferometric Scattering microscopy, have allowed recording of long, uninterrupted single particle trajectories at kilohertz framerates. The resulting data, where particles are continuously detected and do not displace much between observations, thereby do not require complex linking algorithms. Moreover, while these measurements offer more details into the short-term diffusion behaviour of the tracked particles, they are also subject to the influence of localisation uncertainties, which are often underestimated by conventional analysis pipelines. we thus developed a Python library, under the name of TRAIT2D (Tracking Analysis Toolbox – 2D version), in order to track particle diffusion at high sampling rates, and analyse the resulting trajectories with an innovative approach. The data analysis pipeline introduced is more localisation-uncertainty aware, and also selects the most appropriate diffusion model for the data provided on a statistical basis. A trajectory simulation platform also allows the user to handily generate trajectories and even synthetic time-lapses to test alternative tracking algorithms and data analysis approaches. A high degree of customisation for the analysis pipeline, for example with the introduction of different diffusion modes, is possible from the source code. Finally, the presence of graphical user interfaces lowers the access barrier for users with little to no programming experience.
8
Reina, Francesco, John M. A. Wigg, Mariia Dmitrieva, Bela Vogler, Joël Lefebvre, Jens Rittscher, and Christian Eggeling. "TRAIT2D: a Software for Quantitative Analysis of Single Particle Diffusion Data." F1000Research 10 (January 31, 2022): 838. http://dx.doi.org/10.12688/f1000research.54788.2.
Анотація:
Single particle tracking (SPT) is one of the most widely used tools in optical microscopy to evaluate particle mobility in a variety of situations, including cellular and model membrane dynamics. Recent technological developments, such as Interferometric Scattering microscopy, have allowed recording of long, uninterrupted single particle trajectories at kilohertz framerates. The resulting data, where particles are continuously detected and do not displace much between observations, thereby do not require complex linking algorithms. Moreover, while these measurements offer more details into the short-term diffusion behaviour of the tracked particles, they are also subject to the influence of localisation uncertainties, which are often underestimated by conventional analysis pipelines. we thus developed a Python library, under the name of TRAIT2D (Tracking Analysis Toolbox – 2D version), in order to track particle diffusion at high sampling rates, and analyse the resulting trajectories with an innovative approach. The data analysis pipeline introduced is more localisation-uncertainty aware, and also selects the most appropriate diffusion model for the data provided on a statistical basis. A trajectory simulation platform also allows the user to handily generate trajectories and even synthetic time-lapses to test alternative tracking algorithms and data analysis approaches. A high degree of customisation for the analysis pipeline, for example with the introduction of different diffusion modes, is possible from the source code. Finally, the presence of graphical user interfaces lowers the access barrier for users with little to no programming experience.
9
Shin, Kyujin, Yo Song, Yeongchang Goh, and Kang Lee. "Two-Dimensional and Three-Dimensional Single Particle Tracking of Upconverting Nanoparticles in Living Cells." International Journal of Molecular Sciences 20, no. 6 (March 21, 2019): 1424. http://dx.doi.org/10.3390/ijms20061424.
Анотація:
Lanthanide-doped upconversion nanoparticles (UCNPs) are inorganic nanomaterials in which the lanthanide cations embedded in the host matrix can convert incident near-infrared light to visible or ultraviolet light. These particles are often used for long-term and real-time imaging because they are extremely stable even when subjected to continuous irradiation for a long time. It is now possible to image their movement at the single particle level with a scale of a few nanometers and track their trajectories as a function of time with a scale of a few microseconds. Such UCNP-based single-particle tracking (SPT) technology provides information about the intracellular structures and dynamics in living cells. Thus far, most imaging techniques have been built on fluorescence microscopic techniques (epifluorescence, total internal reflection, etc.). However, two-dimensional (2D) images obtained using these techniques are limited in only being able to visualize those on the focal planes of the objective lens. On the contrary, if three-dimensional (3D) structures and dynamics are known, deeper insights into the biology of the thick cells and tissues can be obtained. In this review, we introduce the status of the fluorescence imaging techniques, discuss the mathematical description of SPT, and outline the past few studies using UCNPs as imaging probes or biologically functionalized carriers.
10
Parrish, Emmabeth, Katie A. Rose, Matteo Cargnello, Christopher B. Murray, Daeyeon Lee, and Russell J. Composto. "Nanoparticle diffusion during gelation of tetra poly(ethylene glycol) provides insight into nanoscale structural evolution." Soft Matter 16, no. 9 (2020): 2256–65. http://dx.doi.org/10.1039/c9sm02192b.
Анотація:
Single particle tracking (SPT) of PEG grafted nanoparticles (NPs) was used to examine the gelation of tetra poly(ethylene glycol) (TPEG) succinimidyl glutarate (TPEG-SG) and amine (TPEG-A) terminated 4-armed stars.
11
Godoy, Boris I., Nicholas A. Vickers, and Sean B. Andersson. "An Estimation Algorithm for General Linear Single Particle Tracking Models with Time-Varying Parameters." Molecules 26, no. 4 (February 8, 2021): 886. http://dx.doi.org/10.3390/molecules26040886.
Анотація:
Single Particle Tracking (SPT) is a powerful class of methods for studying the dynamics of biomolecules inside living cells. The techniques reveal the trajectories of individual particles, with a resolution well below the diffraction limit of light, and from them the parameters defining the motion model, such as diffusion coefficients and confinement lengths. Most existing algorithms assume these parameters are constant throughout an experiment. However, it has been demonstrated that they often vary with time as the tracked particles move through different regions in the cell or as conditions inside the cell change in response to stimuli. In this work, we propose an estimation algorithm to determine time-varying parameters of systems that discretely switch between different linear models of motion with Gaussian noise statistics, covering dynamics such as diffusion, directed motion, and Ornstein–Uhlenbeck dynamics. Our algorithm consists of three stages. In the first stage, we use a sliding window approach, combined with Expectation Maximization (EM) to determine maximum likelihood estimates of the parameters as a function of time. These results are only used to roughly estimate the number of model switches that occur in the data to guide the selection of algorithm parameters in the second stage. In the second stage, we use Change Detection (CD) techniques to identify where the models switch, taking advantage of the off-line nature of the analysis of SPT data to create non-causal algorithms with better precision than a purely causal approach. Finally, we apply EM to each set of data between the change points to determine final parameter estimates. We demonstrate our approach using experimental data generated in the lab under controlled conditions.
12
Kim, Jaehi, Sunray Lee, Yeon Kyung Lee, Bomi Seong, Hyung-Mo Kim, San Kyeong, Wooyeon Kim, et al. "In Vitro Tracking of Human Umbilical Vein Endothelial Cells Using Ultra-Sensitive Quantum Dot-Embedded Silica Nanoparticles." International Journal of Molecular Sciences 24, no. 6 (March 17, 2023): 5794. http://dx.doi.org/10.3390/ijms24065794.
Анотація:
The nanoscale spatiotemporal resolution of single-particle tracking (SPT) renders it a powerful method for exploring single-molecule dynamics in living cells or tissues, despite the disadvantages of using traditional organic fluorescence probes, such as the weak fluorescent signal against the strong cellular autofluorescence background coupled with a fast-photobleaching rate. Quantum dots (QDs), which enable tracking targets in multiple colors, have been proposed as an alternative to traditional organic fluorescence dyes; however, they are not ideally suitable for applying SPT due to their hydrophobicity, cytotoxicity, and blinking problems. This study reports an improved SPT method using silica-coated QD-embedded silica nanoparticles (QD2), which represent brighter fluorescence and are less toxic than single QDs. After treatment of QD2 in 10 μg/mL, the label was retained for 96 h with 83.76% of labeling efficiency, without impaired cell function such as angiogenesis. The improved stability of QD2 facilitates the visualization of in situ endothelial vessel formation without real-time staining. Cells retain QD2 fluorescence signal for 15 days at 4 °C without significant photobleaching, indicating that QD2 has overcome the limitations of SPT enabling long-term intracellular tracking. These results proved that QD2 could be used for SPT as a substitute for traditional organic fluorophores or single quantum dots, with its photostability, biocompatibility, and superior brightness.
13
Lee, Yerim, Carey Phelps, Tao Huang, Barmak Mostofian, Daniel Zuckerman, and Xiaolin Nan. "Probing Membrane Nanodomain Organization with Single Particle Tracking via Photoactivated Localization Microscopy (spt-PALM)." Microscopy and Microanalysis 25, S2 (August 2019): 1248–49. http://dx.doi.org/10.1017/s1431927619006974.
14
Patel, Mohak, and Christian Franck. "Position Based Single Particle Tracking (P-SPT) for High-Resolution Deformation and Motion Capture." Biophysical Journal 112, no. 3 (February 2017): 294a. http://dx.doi.org/10.1016/j.bpj.2016.11.1593.
15
Okamoto, Yoshiaki, Seiji Iwasa, and Ryugo Tero. "Quenching Efficiency of Quantum Dots Conjugated to Lipid Bilayers on Graphene Oxide Evaluated by Fluorescence Single Particle Tracking." Applied Sciences 12, no. 8 (April 7, 2022): 3733. http://dx.doi.org/10.3390/app12083733.
Анотація:
A single particle observation of quantum dots (QDs) was performed on lipid bilayers formed on graphene oxide (GO). The long-range fluorescence quenching of GO has been applied to biosensing for various biomolecules. We demonstrated the single particle observation of a QD on supported lipid bilayers in this study, aiming to detect the quenching efficiency of lipid and protein molecules in a lipid bilayer by fluorescence single particle tacking (SPT). A single lipid bilayer or double lipid bilayers were formed on GO flakes deposited on a thermally oxidized silicon substrate by the vesicle fusion method. The QDs were conjugated on the lipid bilayers, and single particle images of the QDs were obtained under the quenching effect of GO. The quenching efficiency of a single QD was evaluated from the fluorescence intensities on the regions with and without GO. The quenching efficiency reflecting the layer numbers of the lipid bilayers was obtained.
16
Saxton, Michael J. "Immobilization in single-particle tracking (SPT): escape from the point spread function (PSF) of the microscope." Biophysical Journal 121, no. 3 (February 2022): 530a. http://dx.doi.org/10.1016/j.bpj.2021.11.2791.
17
Pinholt, Henrik D., Søren S. R. Bohr, Josephine F. Iversen, Wouter Boomsma, and Nikos S. Hatzakis. "Single-particle diffusional fingerprinting: A machine-learning framework for quantitative analysis of heterogeneous diffusion." Proceedings of the National Academy of Sciences 118, no. 31 (July 28, 2021): e2104624118. http://dx.doi.org/10.1073/pnas.2104624118.
Анотація:
Single-particle tracking (SPT) is a key tool for quantitative analysis of dynamic biological processes and has provided unprecedented insights into a wide range of systems such as receptor localization, enzyme propulsion, bacteria motility, and drug nanocarrier delivery. The inherently complex diffusion in such biological systems can vary drastically both in time and across systems, consequently imposing considerable analytical challenges, and currently requires an a priori knowledge of the system. Here we introduce a method for SPT data analysis, processing, and classification, which we term “diffusional fingerprinting.” This method allows for dissecting the features that underlie diffusional behavior and establishing molecular identity, regardless of the underlying diffusion type. The method operates by isolating 17 descriptive features for each observed motion trajectory and generating a diffusional map of all features for each type of particle. Precise classification of the diffusing particle identity is then obtained by training a simple logistic regression model. A linear discriminant analysis generates a feature ranking that outputs the main differences among diffusional features, providing key mechanistic insights. Fingerprinting operates by both training on and predicting experimental data, without the need for pretraining on simulated data. We found this approach to work across a wide range of simulated and experimentally diverse systems, such as tracked lipases on fat substrates, transcription factors diffusing in cells, and nanoparticles diffusing in mucus. This flexibility ultimately supports diffusional fingerprinting’s utility as a universal paradigm for SPT diffusional analysis and prediction.
18
Kæstel-Hansen, Jacob, Tom Kirchhausen, and Nikos S. Hatzakis. "Deep-SPT, a deep learning toolbox for single particle tracking in 3D, reveals how biological motion encodes function." Biophysical Journal 123, no. 3 (February 2024): 43a—44a. http://dx.doi.org/10.1016/j.bpj.2023.11.343.
19
Kodippili, Gayani C., Jeff Spector, Caitlin Sullivan, Richard Labotka, Ken Ritchie, and Philip S. Low. "Evaluation of the Spectrin Compartment Size in Normal and Diseased Red Blood Cells by Single Particle Tracking." Blood 110, no. 11 (November 16, 2007): 143. http://dx.doi.org/10.1182/blood.v110.11.143.143.
Анотація:
Abstract In a process termed “hop diffusion”, membrane proteins diffuse randomly within a cytoskeletally-enclosed compartment over short time scales, periodically hopping from one compartment to the next. Band 3 diffuses in the erythrocyte membrane bilayer in a manner that is thought to be restricted by the size and stability of the spectrin network. Although a fraction of band 3 may exhibit reduced mobility due to its association with spectrin (mediated by proteins such as ankyrin and adducin), another subpopulation of band 3 is thought to diffuse freely within the spectrin compartments, hopping from one corral to the next by transient opening of the barrier (presumably a spectrin tetramer) separating adjacent corrals. Based on this hypothesis, measurement of the compartment size and hopping frequency of a band 3 molecule in the erythrocyte membrane can provide information on the structure and stability of the spectrin network. We have combined single particle tracking (SPT) techniques with high-speed video microscopy to determine the average compartment size and diffusion coefficient of the mobile fraction of band 3 in both normal and diseased red blood cells (RBCs). Streptavidin-linked quantum dots (525nm emission) and 40 nm fluorescent beads were attached to band 3 via a DIDS-biotin-linker, and fluorescence microscopy coupled to a high speed camera was used to determine the position of band 3 with a time resolution of 8 ms (120 fps). Labeling specificity was demonstrated by blotting with streptavidin-horseradish peroxidase, and flow cytometry studies established the binding constant of DIDS-biotin for band 3 in the intact cell of ∼1-2*10−5 M. For diffusion studies, RBCs were incubated with 10−11 M conjugate in order to attach only one or two streptavidin-quantum dots per cell. SPT data were collected on both healthy and pathologic human RBCs, including HbSS (sickle cells), HbSC (sickle cell hemoglobin C), HbSBo (sickle cell zero-beta-thalasamia), HbSB+ (sickle cell beta-plus-thalasamia), HS (hereditary spherocytosis), and HPP (hereditary pyropoikilocytosis). Movement within the observed compartments was found to be random, with an average diagonal length for the mobile fraction in healthy cells of ∼100nm and a diffusion coefficient of ∼1–2*10−10cm2/s. HbSS cells, however, displayed a comparatively smaller compartment size (∼60–80nm) with a lower diffusion coefficient (0.1−1*10−10cm2/s). In contrast, HPP cells exhibited an average compartment size of ∼133nm with a diffusion coefficient of 6*10−10cm2/s. SPT data further suggest the presence of multiple populations of band 3, exhibiting both free diffusion and restricted mobilities with different characteristics. Details of band 3 diffusion in all of the aforementioned diseased and normal blood cells will be provided, and a model accounting for the multiple populations of band 3 will be presented.
20
Das, Sulagna, Taofei Yin, Qingfen Yang, Jingqiao Zhang, Yi I. Wu, and Ji Yu. "Single-molecule tracking of small GTPase Rac1 uncovers spatial regulation of membrane translocation and mechanism for polarized signaling." Proceedings of the National Academy of Sciences 112, no. 3 (January 5, 2015): E267—E276. http://dx.doi.org/10.1073/pnas.1409667112.
Анотація:
Polarized Rac1 signaling is a hallmark of many cellular functions, including cell adhesion, motility, and cell division. The two steps of Rac1 activation are its translocation to the plasma membrane and the exchange of nucleotide from GDP to GTP. It is, however, unclear whether these two processes are regulated independent of each other and what their respective roles are in polarization of Rac1 signaling. We designed a single-particle tracking (SPT) method to quantitatively analyze the kinetics of Rac1 membrane translocation in living cells. We found that the rate of Rac1 translocation was significantly elevated in protrusions during cell spreading on collagen. Furthermore, combining FRET sensor imaging with SPT measurements in the same cell, the recruitment of Rac1 was found to be polarized to an extent similar to that of the nucleotide exchange process. Statistical analysis of single-molecule trajectories and optogenetic manipulation of membrane lipids revealed that Rac1 membrane translocation precedes nucleotide exchange, and is governed primarily by interactions with phospholipids, particularly PI(3,4,5)P3, instead of protein factors. Overall, the study highlights the significance of membrane translocation in spatial Rac1 signaling, which is in addition to the traditional view focusing primarily on GEF distribution and exchange reaction.
21
Li, Yidi, Hong Liu, Bing Yang, Runwei Ding, and Yang Chen. "Deep Metric Learning-Assisted 3D Audio-Visual Speaker Tracking via Two-Layer Particle Filter." Complexity 2020 (August 31, 2020): 1–8. http://dx.doi.org/10.1155/2020/3764309.
Анотація:
For speaker tracking, integrating multimodal information from audio and video provides an effective and promising solution. The current challenges are focused on the construction of a stable observation model. To this end, we propose a 3D audio-visual speaker tracker assisted by deep metric learning on the two-layer particle filter framework. Firstly, the audio-guided motion model is applied to generate candidate samples in the hierarchical structure consisting of an audio layer and a visual layer. Then, a stable observation model is proposed with a designed Siamese network, which provides the similarity-based likelihood to calculate particle weights. The speaker position is estimated using an optimal particle set, which integrates the decisions from audio particles and visual particles. Finally, the long short-term mechanism-based template update strategy is adopted to prevent drift during tracking. Experimental results demonstrate that the proposed method outperforms the single-modal trackers and comparison methods. Efficient and robust tracking is achieved both in 3D space and on image plane.
22
Durso, William, Manuella Martins, Laura Marchetti, Federico Cremisi, Stefano Luin, and Francesco Cardarelli. "Lysosome Dynamic Properties during Neuronal Stem Cell Differentiation Studied by Spatiotemporal Fluctuation Spectroscopy and Organelle Tracking." International Journal of Molecular Sciences 21, no. 9 (May 11, 2020): 3397. http://dx.doi.org/10.3390/ijms21093397.
Анотація:
We investigated lysosome dynamics during neuronal stem cell (NSC) differentiation by two quantitative and complementary biophysical methods based on fluorescence: imaging-derived mean square displacement (iMSD) and single-particle tracking (SPT). The former extracts the average dynamics and size of the whole population of moving lysosomes directly from imaging, with no need to calculate single trajectories; the latter resolves the finest heterogeneities and dynamic features at the single-lysosome level, which are lost in the iMSD analysis. In brief, iMSD analysis reveals that, from a structural point of view, lysosomes decrement in size during NSC differentiation, from 1 μm average diameter in the embryonic cells to approximately 500 nm diameter in the fully differentiated cells. Concomitantly, iMSD analysis highlights modification of key dynamic parameters, such as the average local organelle diffusivity and anomalous coefficient, which may parallel cytoskeleton remodeling during the differentiation process. From average to local, SPT allows mapping heterogeneous dynamic responses of single lysosomes in different districts of the cells. For instance, a dramatic decrease of lysosomal transport in the soma is followed by a rapid increase of transport in the projections at specific time points during neuronal differentiation, an observation compatible with the hypothesis that lysosomal active mobilization shifts from the soma to the newborn projections. Our combined results provide new insight into the lysosome size and dynamics regulation throughout NSC differentiation, supporting new functions proposed for this organelle.
23
DE KEERSMAECKER, H., S. ROCHA, E. FRON, H. UJI-I, J. HOFKENS, and H. MIZUNO. "EGF RECEPTOR DYNAMICS IN EGF-RESPONDING CELLS REVEALED BY FUNCTIONAL IMAGING DURING SINGLE PARTICLE TRACKING." Biophysical Reviews and Letters 08, no. 03n04 (December 2013): 229–42. http://dx.doi.org/10.1142/s1793048013500070.
Анотація:
The epidermal growth factor (EGF) receptor transduces the extracellular EGF signal into the cells. The distribution of these EGF receptors in the plasma membrane is heterogeneous and dynamic, which is proposed to be important for the regulation of cell signaling. The response of the cells to a physiological concentration of EGF is not homogeneous, which makes it difficult to analyze the dynamics related to the response. Here we developed a system to perform functional imaging during single particle tracking (SPT) analysis. This system made it possible to observe the cytosolic Ca 2+ concentration to monitor the cell response while tracking individual EGF molecules and found that about half of the cells responded to the stimulation with 1.6 nM EGF. In the responding cells, the EGF receptor showed 3 modes of movement: fast (the diffusion coefficient of 0.081 ± 0.009 μm2/sec, 29 ± 9%), slow (0.020 ± 0.005 μm2/sec, 22 ± 6%), and stationary (49 ± 13%). The diffusion coefficient of the fast mode movement in the responding cells was significantly larger than that in the nonresponding cells (0.069 ± 0.009 μm2/sec, p < 0.05). The diffusion coefficient of the fast mode movement is thought to reflect the monomer–dimer equilibrium of the EGF receptor. We assumed that the feedback regulation via the Ca 2+ signaling pathway slightly shifts the equilibrium from dimer to monomer in the responding cells. [Formula: see text]Special Issue Comment: This research paper is about the diffusion of EGF receptors in the membrane. It is therefore related with various projects in this Special Issue: the reviews about FRET41 and enzymes,42 and the projects about solving single molecules trajectories.43
24
Iversen, Josephine F., Søren S. R. Bohr, Henrik D. Pinholt, Matias E. Moses, Lars Iversen, Sune M. Christensen, Nikos S. Hatzakis, and Min Zhang. "Single-Particle Tracking of Thermomyces lanuginosus Lipase Reveals How Mutations in the Lid Region Remodel Its Diffusion." Biomolecules 13, no. 4 (March 31, 2023): 631. http://dx.doi.org/10.3390/biom13040631.
Анотація:
The function of most lipases is controlled by the lid, which undergoes conformational changes at a water–lipid interface to expose the active site, thus activating catalysis. Understanding how lid mutations affect lipases’ function is important for designing improved variants. Lipases’ function has been found to correlate with their diffusion on the substrate surface. Here, we used single-particle tracking (SPT), a powerful tool for deciphering enzymes’ diffusional behavior, to study Thermomyces lanuginosus lipase (TLL) variants with different lid structures in a laundry-like application condition. Thousands of parallelized recorded trajectories and hidden Markov modeling (HMM) analysis allowed us to extract three interconverting diffusional states and quantify their abundance, microscopic transition rates, and the energy barriers for sampling them. Combining those findings with ensemble measurements, we determined that the overall activity variation in the application condition is dependent on surface binding and lipase mobility when bound. Specifically, the L4 variant with a TLL-like lid and wild-type (WT) TLL displayed similar ensemble activity, but WT bound stronger to the surface than L4, while L4 had a higher diffusion coefficient and thus activity when bound to the surface. These mechanistic elements can only be de-convoluted by our combined assays. Our findings offer fresh perspectives on the development of the next iteration of enzyme-based detergent.
25
Gu, Yeyi, Xinmin Zhou, Minjie Wan, and Guohua Gu. "Visual Target Tracking using Robust Information Interaction between Single Tracker and Online Model." MATEC Web of Conferences 232 (2018): 02046. http://dx.doi.org/10.1051/matecconf/201823202046.
Анотація:
In this paper, a novel tracking algorithm based on the cooperative operation of online appearance model and typical tracking in contiguous frames is proposed. First of all, to achieve satisfactory performances in challenging scenes, we focus on establishing a robust discriminative tracking model with linear Support Vector Machine (SVM) and use the particle filter for localization. Intended to fit the particle filter, the outputs of SVM classifier are mapped into probabilities with a sigmoid function so that the posterior of candidate samples is estimated. Then, the tracking loop starts with median flow method and the coordinated operation of the two trackers is mediated by the maximum a posteriori (MAP) estimate for the target probability of negative samples, which is defined during the sigmoid fit. Lastly, for the purpose of model update, we sum up the optimal SVM using a prototype set with the predefined budget, and the classifier is updated on both the prototype set and the updated data from the tracking results every few frames. A number of comparative experiments are conducted on real video sequences and both qualitative and quantitative evaluations demonstrate a robust and precise performance of our method.
26
Shaevitz, Joshua W. "Bayesian Estimation of the Axial Position in Astigmatism-Based Three-Dimensional Particle Tracking." International Journal of Optics 2009 (2009): 1–6. http://dx.doi.org/10.1155/2009/896208.
Анотація:
Accurate estimation of the axial position of a molecule using a single lateral image remains a challenge in fluorescent single particle tracking. Here, a principled algorithm for the Bayesian estimation of the axial position of a molecule in three-dimensional astigmatism-based particle tracking is proposed. This technique uses the data from a calibration image set to derive the position without assuming a functional form for the abberated defocusing curve. Using a calibration image set from forty 57 nm beads, the axial position is calculated, and the error associated with position estimation is discussed. This method is compared to previously published algorithms.
27
Ikoma, Norikazu, Ryuichi Yamaguchi, Hideaki Kawano, and Hiroshi Maeda. "Tracking of Multiple Moving Objects in Dynamic Image of Omni-Directional Camera Using PHD Filter." Journal of Advanced Computational Intelligence and Intelligent Informatics 12, no. 1 (January 20, 2008): 16–25. http://dx.doi.org/10.20965/jaciii.2008.p0016.
Анотація:
A method of multiple moving objects tracking in dynamic image of omni-directional camera has been proposed. Finite random set (FRS) based state space model is employed in the method due to its inherent nature capable to represent the scene having occlusion and appearance of object as well as missing and false detection in observation. Sequential Monte Carlo (SMC) implementation of Probability hypothesis density (PHD) filter has been used for estimating state of the state space model. The state is a finite random set of single object states, where each element of the set consists of position and velocity of the object in panoramic image coordinate of omni-directional camera image. We propose a new method to display tracking result from weighted particles obtained from the estimation process by SMC implementation of PHD filter. Key idea of the method is to put an integer label on each particle, where the label indicates specific object among multiple objects in the image scene tracked by the particle. Numerical simulation and real image experiments illustrate tracking performance of the proposed method.
28
Lin, Ye, and Sean B. Andersson. "Expectation maximization based framework for joint localization and parameter estimation in single particle tracking from segmented images." PLOS ONE 16, no. 5 (May 21, 2021): e0243115. http://dx.doi.org/10.1371/journal.pone.0243115.
Анотація:
Single Particle Tracking (SPT) is a well known class of tools for studying the dynamics of biological macromolecules moving inside living cells. In this paper, we focus on the problem of localization and parameter estimation given a sequence of segmented images. In the standard paradigm, the location of the emitter inside each frame of a sequence of camera images is estimated using, for example, Gaussian fitting (GF), and these locations are linked to provide an estimate of the trajectory. Trajectories are then analyzed by using Mean Square Displacement (MSD) or Maximum Likelihood Estimation (MLE) techniques to determine motion parameters such as diffusion coefficients. However, the problems of localization and parameter estimation are clearly coupled. Motivated by this, we have created an Expectation Maximization (EM) based framework for simultaneous localization and parameter estimation. We demonstrate this framework through two representative methods, namely, Sequential Monte Carlo combined with Expectation Maximization (SMC-EM) and Unscented Kalman Filter combined with Expectation Maximization (U-EM). Using diffusion in two-dimensions as a prototypical example, we conduct quantitative investigations on localization and parameter estimation performance across a wide range of signal to background ratios and diffusion coefficients and compare our methods to the standard techniques based on GF-MSD/MLE. To demonstrate the flexibility of the EM based framework, we do comparisons using two different camera models, an ideal camera with Poisson distributed shot noise but no readout noise, and a camera with both shot noise and the pixel-dependent readout noise that is common to scientific complementary metal-oxide semiconductor (sCMOS) camera. Our results indicate our EM based methods outperform the standard techniques, especially at low signal levels. While U-EM and SMC-EM have similar accuracy, U-EM is significantly more computationally efficient, though the use of the Unscented Kalman Filter limits U-EM to lower diffusion rates.
29
Sako, Yasushi, Akira Nagafuchi, Shoichiro Tsukita, Masatoshi Takeichi, and Akihiro Kusumi. "Cytoplasmic Regulation of the Movement of E-Cadherin on the Free Cell Surface as Studied by Optical Tweezers and Single Particle Tracking: Corralling and Tethering by the Membrane Skeleton." Journal of Cell Biology 140, no. 5 (March 9, 1998): 1227–40. http://dx.doi.org/10.1083/jcb.140.5.1227.
Анотація:
The translational movement of E-cadherin, a calcium-dependent cell–cell adhesion molecule in the plasma membrane in epithelial cells, and the mechanism of its regulation were studied using single particle tracking (SPT) and optical tweezers (OT). The wild type (Wild) and three types of artificial cytoplasmic mutants of E-cadherin were expressed in L-cells, and their movements were compared. Two mutants were E-cadherins that had deletions in the COOH terminus and lost the catenin-binding site(s) in the COOH terminus, with remaining 116 and 21 amino acids in the cytoplasmic domain (versus 152 amino acids for Wild); these are called Catenin-minus and Short-tailed in this paper, respectively. The third mutant, called Fusion, is a fusion protein between E-cadherin without the catenin-binding site and α-catenin without its NH2-terminal half. These cadherins were labeled with 40-nm φ colloidal gold or 210-nm φ latex particles via a monoclonal antibody to the extracellular domain of E-cadherin for SPT or OT experiments, respectively. E-cadherin on the dorsal cell surface (outside the cell–cell contact region) was investigated. Catenin-minus and Short-tailed could be dragged an average of 1.1 and 1.8 μm by OT (trapping force of 0.8 pN), and exhibited average microscopic diffusion coefficients (Dmicro) of 1.2 × 10−10 and 2.1 × 10−10 cm2/s, respectively. Approximately 40% of Wild, Catenin-minus, and Short-tailed exhibited confined-type diffusion. The confinement area was 0.13 μm2 for Wild and Catenin-minus, while that for Short-tailed was greater by a factor of four. In contrast, Fusion could be dragged an average of only 140 nm by OT. Average Dmicro for Fusion measured by SPT was small (0.2 × 10−10 cm2/s). These results suggest that Fusion was bound to the cytoskeleton. Wild consists of two populations; about half behaves like Catenin- minus, and the other half behaves like Fusion. It is concluded that the movements of the wild-type E-cadherin in the plasma membrane are regulated via the cytoplasmic domain by (a) tethering to actin filaments through catenin(s) (like Fusion) and (b) a corralling effect of the network of the membrane skeleton (like Catenin-minus). The effective spring constants of the membrane skeleton that contribute to the tethering and corralling effects as measured by the dragging experiments were 30 and 5 pN/μm, respectively, indicating a difference in the skeletal structures that produce these two effects.
30
González-Domínguez, Irene, Eduard Puente-Massaguer, Laura Cervera, and Francesc Gòdia. "Quality Assessment of Virus-Like Particles at Single Particle Level: A Comparative Study." Viruses 12, no. 2 (February 17, 2020): 223. http://dx.doi.org/10.3390/v12020223.
Анотація:
Virus-like particles (VLPs) have emerged as a powerful scaffold for antigen presentation and delivery strategies. Compared to single protein-based therapeutics, quality assessment requires a higher degree of refinement due to the structure of VLPs and their similar properties to extracellular vesicles (EVs). Advances in the field of nanotechnology with single particle and high-resolution analysis techniques provide appealing approaches to VLP characterization. In this study, six different biophysical methods have been assessed for the characterization of HIV-1-based VLPs produced in mammalian and insect cell platforms. Sample preparation and equipment set-up were optimized for the six strategies evaluated. Electron Microscopy (EM) disclosed the presence of several types of EVs within VLP preparations and cryogenic transmission electron microscopy (cryo-TEM) resulted in the best technique to resolve the VLP ultrastructure. The use of super-resolution fluorescence microscopy (SRFM), nanoparticle tracking analysis (NTA) and flow virometry enabled the high throughput quantification of VLPs. Interestingly, differences in the determination of nanoparticle concentration were observed between techniques. Moreover, NTA and flow virometry allowed the quantification of both EVs and VLPs within the same experiment while analyzing particle size distribution (PSD), simultaneously. These results provide new insights into the use of different analytical tools to monitor the production of nanoparticle-based biologicals and their associated contaminants.
31
Woringer, Maxime, and Xavier Darzacq. "Protein motion in the nucleus: from anomalous diffusion to weak interactions." Biochemical Society Transactions 46, no. 4 (July 31, 2018): 945–56. http://dx.doi.org/10.1042/bst20170310.
Анотація:
Understanding how transcription factors (TFs) regulate mammalian gene expression in space and time is a central topic in biology. To activate a gene, a TF has first to diffuse in the available space of the nucleus until it reaches a target DNA sequence or protein (target site). This eventually results in the recruitment of the whole transcriptional machinery. All these processes take place in the mammalian nucleoplasm, a highly organized and dynamic environment, in which some complexes transiently assemble and break apart, whereas others appear more stable. This diversity of dynamic behaviors arises from the number of biomolecules that make up the nucleoplasm and their pairwise interactions. Indeed, interactions energies that span several orders of magnitude, from covalent bounds to transient and dynamic interactions, can shape nuclear landscapes. Thus, the nuclear environment determines how frequently and how fast a TF contacts its target site, and it indirectly regulates gene expression. How exactly transient interactions are involved in the regulation of TF diffusion is unclear, but are reflected by live cell imaging techniques, including single-particle tracking (SPT). Overall, the macroscopic result of these microscopic interactions is almost always anomalous diffusion, a phenomenon widely studied and modeled. Here, we review the connections between the anomalous diffusion of a TF observed by SPT and the microscopic organization of the nucleus, including recently described topologically associated domains and dynamic phase-separated compartments. We propose that anomalous diffusion found in SPT data result from weak and transient interactions with dynamic nuclear substructures, and that SPT data analysis would benefit from a better description of such structures.
32
Costello, Deirdre A., Gary R. Whittaker, and Susan Daniel. "Variations in pH Sensitivity, Acid Stability, and Fusogenicity of Three Influenza Virus H3 Subtypes." Journal of Virology 89, no. 1 (October 15, 2014): 350–60. http://dx.doi.org/10.1128/jvi.01927-14.
Анотація:
ABSTRACTInfluenza A virus strains adapt to achieve successful entry into host species. Entry is mediated by the viral membrane protein hemagglutinin (HA), which triggers membrane fusion and genome release under acidic conditions in the endosome. In addition to changes in the receptor binding domain, the acid stability of HA has been linked to the successful transmission of virus between avian and human hosts. However, to fully understand the connection between changes in HA and host tropism, additional factors relevant to HA structure-function and membrane fusion are also likely to be important. Using single-particle-tracking (SPT) techniques, individual membrane fusion events can be observed under specific conditions, which provide detailed information regarding HA pH sensitivity and acid stability and the rate and extent of membrane fusion. This provides a comparative way to characterize and distinguish influenza virus fusion properties among virus strains. We used SPT to quantify the fusion properties of three H3 influenza strains: A/Aichi/68/H3N2 (X:31), A/Udorn/72/H3N2 (Udorn), and A/Brisbane/07/H3N2 (Brisbane). The rate of fusion for the most clinically relevant strain, Brisbane, is generally insensitive to decreasing pH, while the fusion of the egg-adapted strains Udorn and X:31 is strongly dependent on pH (and is faster) as the pH decreases. All strains exhibit similar acid stability (the length of time that they remain fusogenic in an acidic environment) at higher pHs, but the egg-adapted strains become less acid stable at lower pHs. Thus, it appears that the laboratory-adapted H3 strains tested may have evolved to compensate for the faster HA deactivation at low pH, with a commensurate increase in the rate of fusion and number of proteins facilitating fusion, relative to the Brisbane strain.IMPORTANCEThe ability of influenza virus to release its genome under different acidic conditions has recently been linked to the transmission of influenza virus between different species. However, it is yet to be determined how acid-induced membrane fusion varies with virus strain and influences tropism. The results presented here are the results of an intra-H3-subtype study of acid stability and fusion kinetics. Using a single-particle-tracking (SPT) technique, we show here that the highest pH that initiates fusion is not necessarily the pH at which the kinetics of fusion is fastest and most abundant for a given strain. Strains exhibit different fusion behaviors, as evidenced by their unique kinetic trends; pH sensitivities, as evidenced by the differences when the first fusion events commence; and HA stabilities, as evidenced by the length of time that virions can persist in an acidic environment and still be fusion competent.
33
Cosetta, Ravelli, Corsini Michela, Ventura Anna, Domenichini Mattia, Grillo Elisabetta, and Mitola Stefania. "Alternative method to visualize receptor dynamics in cell membranes." PLOS ONE 19, no. 6 (June 11, 2024): e0304172. http://dx.doi.org/10.1371/journal.pone.0304172.
Анотація:
There is a close relation between membrane receptor dynamics and their behavior. Several microscopy techniques have been developed to study protein dynamics in live cells such as the Fluorescence Recovery After Photobleaching (FRAP) or the Single Particle Tracking (SPT). These methodologies require expensive instruments, are time consuming, allow the analysis of small portion of the cell or an extremely small number of receptors at a time. Here we propose a time-saving approach that allows to visualize the entire receptor pool and its localization in time. This protocol requires an epifluorescence microscope equipped for structured illuminated sectioning and for live cell imaging. It can be applied to characterize membrane receptor and multi protein complex and their response to activators or inhibitors. Image acquisition and analysis can be performed in two days, while cells and substratum preparation require a few minutes a day for three days.
34
COLE, CHRISTINE LIND, and HONG QIAN. "THE BROWNIAN RATCHET REVISITED: DIFFUSION FORMALISM, POLYMER-BARRIER ATTRACTIONS, AND MULTIPLE FILAMENTOUS BUNDLE GROWTH." Biophysical Reviews and Letters 06, no. 01n02 (June 2011): 59–79. http://dx.doi.org/10.1142/s1793048011001269.
Анотація:
Actin polymerization driven stochastic movement of the bacteria Listeria monocytogenes is often measured using single-particle tracking (SPT) methodology and analyzed in terms of statistics. Experimental results suggested a dynamic association between the growing actin filaments and the propelled bacteria. Based on an alternative mathematical formalism for a Brownian ratchet (BR), we introduce such an attractive interaction into the one-dimensional BR model and show that its effect is equivalent to an external resistant force on the bacterium. Such a force significantly reduces the Brownian motion of a driven bacterium, and accentuates the stepping due to polymerization. We then consider the growth, with and without a barrier, of a filamentous bundle consisting of N identical filaments. It is shown that the bundle grows with a similar rate as a single filament in the absence of a load, but can oppose N times the external force under the stalling condition. A set of relationships describing the velocity of the bacterium movement (Vz) and its apparent diffusivity (Dz) as functions of the resistant force (F) and the number of filaments in a bundle (N) are obtained. The theoretical study suggests methods for data analysis in future experiments with applied external resistant force.
35
Motegi, Toshinori, Kingo Takiguchi, Yohko Tanaka-Takiguchi, Toshiki Itoh, and Ryugo Tero. "Physical Properties and Reactivity of Microdomains in Phosphatidylinositol-Containing Supported Lipid Bilayer." Membranes 11, no. 5 (May 3, 2021): 339. http://dx.doi.org/10.3390/membranes11050339.
Анотація:
We characterized the size, distribution, and fluidity of microdomains in a lipid bilayer containing phosphatidylinositol (PI) and revealed their roles during the two-dimensional assembly of a membrane deformation protein (FBP17). The morphology of the supported lipid bilayer (SLB) consisting of PI and phosphatidylcholine (PC) on a mica substrate was observed with atomic force microscope (AFM). Single particle tracking (SPT) was performed for the PI+PC-SLB on the mica substrate by using the diagonal illumination setup. The AFM topography showed that PI-derived submicron domains existed in the PI+PC-SLB. The spatiotemporal dependence of the lateral lipid diffusion obtained by SPT showed that the microdomain had lower fluidity than the surrounding region and worked as the obstacles for the lipid diffusion. We observed the two-dimensional assembly of FBP17, which is one of F-BAR family proteins included in endocytosis processes and has the function generating lipid bilayer tubules in vitro. At the initial stage of the FBP17 assembly, the PI-derived microdomain worked as a scaffold for the FBP17 adsorption, and the fluid surrounding region supplied FBP17 to grow the FBP17 domain via the lateral molecular diffusion. This study demonstrated an example clearly revealing the roles of two lipid microregions during the protein reaction on a lipid bilayer.
36
Yang, Ke, Yuanyuan Wang, Bo Sun, Tian Tian, Zhu Dai, and Zhongdang Xiao. "Dynamics and Traffic for Transfecting Exogenous MicroRNA as Studied by Live-Cell Microscopy." Journal of Biomedical Nanotechnology 17, no. 8 (August 1, 2021): 1647–53. http://dx.doi.org/10.1166/jbn.2021.3129.
Анотація:
MicroRNA (miRNA) has emerged as an important gene-regulator that shows great potential in gene therapy because of its unique roles in gene-regulation. However, the knowledge on their function and transportation in vivo is still lacking, and there are limited obvious evidences to define intracellular transportation of miRNA. In this study, the dynamics of exogenous miR-21 transfected into HeLa cells was traced by live-cell microscopy. Their transportation at key time points was recorded and dynamic properties were analyzed by single particle tracking (SPT) and mean square displacement (MSD) calculation. Results showed that the exogenous miRNAs bounded to cells quickly and went through lysosome into cytosol, where they were subsequently recruited into p-body. They finally were degraded, otherwise went back to cytosol in some way. Long time observation and analysis of motion mode showed that the miRNAs were confined in a small region and their motion modes were flexible in different intracellular microenvironment after entering the cells.
37
Bolaños-Jiménez, Rocío, Massimiliano Rossi, David Fernandez Rivas, Christian J. Kähler, and Alvaro Marin. "Streaming flow by oscillating bubbles: quantitative diagnostics via particle tracking velocimetry." Journal of Fluid Mechanics 820 (May 10, 2017): 529–48. http://dx.doi.org/10.1017/jfm.2017.229.
Анотація:
Oscillating microbubbles can be used as microscopic agents. Using external acoustic fields they are able to set the surrounding fluid into motion, erode surfaces and even to carry particles attached to their interfaces. Although the acoustic streaming flow that the bubble generates in its vicinity has been often observed, it has never been measured and quantitatively compared with the available theoretical models. The scarcity of quantitative data is partially due to the strong three-dimensional character of bubble-induced streaming flows, which demands advanced velocimetry techniques. In this work, we present quantitative measurements of the flow generated by single and pairs of acoustically excited sessile microbubbles using a three-dimensional particle tracking technique. Using this novel experimental approach we are able to obtain the bubble’s resonant oscillating frequency, study the boundaries of the linear oscillation regime, give predictions on the flow strength and the shear in the surrounding surface and study the flow and the stability of a two-bubble system. Our results show that velocimetry techniques are a suitable tool to make diagnostics on the dynamics of acoustically excited microbubbles.
38
Stasiak, Łukasz A., and Andrzej Pacut. "Face Tracking and Recognition with the Use of Particle-Filtered Local Features." Journal of Telecommunications and Information Technology, no. 4 (June 27, 2023): 26–36. http://dx.doi.org/10.26636/jtit.2010.4.1093.
Анотація:
A consistent particle filtering-based framework for the purpose of parallel face tracking and recognition from video sequences is proposed. A novel approach to defining randomized, particle filtering-driven local face features for the purpose of recognition is proposed. The potential of cumulating classification decisions based on the proposed feature set definition is evaluated. By applying cumulation mechanisms to the classification results determined from single frames and with the use of particle-filtered features, good recognition rates are obtained at the minimal computational cost. The proposed framework can operate in real-time on a typical modern PC. Additionally, the application of cumulation mechanisms makes the framework resistant to brief visual distortions, such as occlusions, head rotations or face expressions. A high performance is also obtained on low resolution images (video frames). Since the framework is based on the particle filtering principle, it is easily tunable to various application requirements (security level, hardware constraints).
39
Imran Sainan, Khairul, Mirwansah Mohamad, Zulkifli Mohamed3, Saiful Bahari Saari, Muhd Faiz Mat, and Nurul Syuhadah Khusaini. "Athletes Tracking using Homography Method: a Preliminary Study." International Journal of Engineering & Technology 7, no. 4.27 (November 30, 2018): 6. http://dx.doi.org/10.14419/ijet.v7i4.27.22427.
Анотація:
Particle tracking has been used widely to track a single particle motion or trajectory in a medium. One of the applications of the particle tracking is in sports analysis. The tracking method can be divided into, the wearable device based system and the image based system. The wearable device based system utilizing global (GPS) and local (LPM) positioning system to track player movement. The image based system use video image processing to track the player movement and recorded is frames basis. However, the image processing method in a football match requires correction as it is normally recorded from the side of the field. Thus, to solve this problem a set of mathematical solution is needed to convert the image coordinate system (pixels) to the actual coordinate system (meters). The most commonly used is the homography method. The technique requires at least 4 reference points to transform the image coordinate into the actual coordinate system. In this project, a futsal game was recorded. The image coordinate of the player were marked in each frame with respect to the time. The image coordinate data were converted into the actual coordinate using homography matrix. Comparisons were made between the homography technique method and open-source available image warp processing method for validation. Based on the result, the homography coordinate transformation system produce a good agreement with actual player activity on the field.
40
Smith, Carlas S., Sjoerd Stallinga, Keith A. Lidke, Bernd Rieger, and David Grunwald. "Probability-based particle detection that enables threshold-free and robust in vivo single-molecule tracking." Molecular Biology of the Cell 26, no. 22 (November 5, 2015): 4057–62. http://dx.doi.org/10.1091/mbc.e15-06-0448.
Анотація:
Single-molecule detection in fluorescence nanoscopy has become a powerful tool in cell biology but can present vexing issues in image analysis, such as limited signal, unspecific background, empirically set thresholds, image filtering, and false-positive detection limiting overall detection efficiency. Here we present a framework in which expert knowledge and parameter tweaking are replaced with a probability-based hypothesis test. Our method delivers robust and threshold-free signal detection with a defined error estimate and improved detection of weaker signals. The probability value has consequences for downstream data analysis, such as weighing a series of detections and corresponding probabilities, Bayesian propagation of probability, or defining metrics in tracking applications. We show that the method outperforms all current approaches, yielding a detection efficiency of >70% and a false-positive detection rate of <5% under conditions down to 17 photons/pixel background and 180 photons/molecule signal, which is beneficial for any kind of photon-limited application. Examples include limited brightness and photostability, phototoxicity in live-cell single-molecule imaging, and use of new labels for nanoscopy. We present simulations, experimental data, and tracking of low-signal mRNAs in yeast cells.
41
Zhuo, Guan-Yu, Ming-Chi Chen, Tzu-Yu Lin, Shih-Ting Lin, Daniel Tzu-Li Chen та Cynthia Wei-Sheng Lee. "Opioid-Modulated Receptor Localization and Erk1/2 Phosphorylation in Cells Coexpressing μ-Opioid and Nociceptin Receptors". International Journal of Molecular Sciences 24, № 2 (5 січня 2023): 1048. http://dx.doi.org/10.3390/ijms24021048.
Анотація:
We attempted to examine the alterations elicited by opioids via coexpressed μ-opioid (MOP) and nociceptin/orphanin FQ (NOP) receptors for receptor localization and Erk1/2 (p44/42 MAPK) in human embryonic kidney (HEK) 293 cells. Through two-photon microscopy, the proximity of MOP and NOP receptors was verified by fluorescence resonance energy transfer (FRET), and morphine but not buprenorphine facilitated the process of MOP-NOP heterodimerization. Single-particle tracking (SPT) further revealed that morphine or buprenorphine hindered the movement of the MOP-NOP heterodimers. After exposure to morphine or buprenorphine, receptor localization on lipid rafts was detected by immunocytochemistry, and phosphorylation of Erk1/2 was determined by immunoblotting in HEK 293 cells expressing MOP, NOP, or MOP+NOP receptors. Colocalization of MOP and NOP on lipid rafts was enhanced by morphine but not buprenorphine. Morphine stimulated the phosphorylation of Erk1/2 with a similar potency in HEK 293 cells expressing MOP and MOP+NOP receptors, but buprenorphine appeared to activate Erk1/2 solely through NOP receptors. Our results suggest that opioids can fine-tune the cellular localization of opioid receptors and phosphorylation of Erk1/2 in MOP+NOP-expressing cells.
42
Sanabria-Macias, Frank, Marta Marron-Romera, and Javier Macias-Guarasa. "Audiovisual Tracking of Multiple Speakers in Smart Spaces." Sensors 23, no. 15 (August 5, 2023): 6969. http://dx.doi.org/10.3390/s23156969.
Анотація:
This paper presents GAVT, a highly accurate audiovisual 3D tracking system based on particle filters and a probabilistic framework, employing a single camera and a microphone array. Our first contribution is a complex visual appearance model that accurately locates the speaker’s mouth. It transforms a Viola & Jones face detector classifier kernel into a likelihood estimator, leveraging knowledge from multiple classifiers trained for different face poses. Additionally, we propose a mechanism to handle occlusions based on the new likelihood’s dispersion. The audio localization proposal utilizes a probabilistic steered response power, representing cross-correlation functions as Gaussian mixture models. Moreover, to prevent tracker interference, we introduce a novel mechanism for associating Gaussians with speakers. The evaluation is carried out using the AV16.3 and CAV3D databases for Single- and Multiple-Object Tracking tasks (SOT and MOT, respectively). GAVT significantly improves the localization performance over audio-only and video-only modalities, with up to 50.3% average relative improvement in 3D when compared with the video-only modality. When compared to the state of the art, our audiovisual system achieves up to 69.7% average relative improvement for the SOT and MOT tasks in the AV16.3 dataset (2D comparison), and up to 18.1% average relative improvement in the MOT task for the CAV3D dataset (3D comparison).
43
Yang, Yantao, Na Qu, Jie Tan, Muaz N. Rushdi, Christopher J. Krueger, and Antony K. Chen. "Roles of Gag-RNA interactions in HIV-1 virus assembly deciphered by single-molecule localization microscopy." Proceedings of the National Academy of Sciences 115, no. 26 (June 11, 2018): 6721–26. http://dx.doi.org/10.1073/pnas.1805728115.
Анотація:
During HIV-1 assembly, the retroviral structural protein Gag forms an immature capsid, containing thousands of Gag molecules, at the plasma membrane (PM). Interactions between Gag nucleocapsid (NC) and viral RNA (vRNA) are thought to drive assembly, but the exact roles of these interactions have remained poorly understood. Since previous studies have shown that Gag dimer- or trimer-forming mutants (GagZiL) lacking an NC domain can form immature capsids independent of RNA binding, it is often hypothesized that vRNA drives Gag assembly by inducing Gag to form low-ordered multimers, but is dispensable for subsequent assembly. In this study, we examined the role of vRNA in HIV-1 assembly by characterizing the distribution and mobility of Gag and Gag NC mutants at the PM using photoactivated localization microscopy (PALM) and single-particle tracking PALM (spt-PALM). We showed that both Gag and GagZiLassembly involve a similar basic assembly unit, as expected. Unexpectedly, the two proteins underwent different subsequent assembly pathways, with Gag cluster density increasing asymptotically, while GagZiLcluster density increased linearly. Additionally, the directed movement of Gag, but not GagZiL, was maintained at a constant speed, suggesting that the two proteins experience different external driving forces. Assembly was abolished when Gag was rendered monomeric by NC deletion. Collectively, these results suggest that, beyond inducing Gag to form low-ordered multimer basic assembly units, vRNA is essential in scaffolding and maintaining the stability of the subsequent assembly process. This finding should advance the current understanding of HIV-1 and, potentially, other retroviruses.
44
Etheridge, Thomas J., Antony M. Carr, and Alex D. Herbert. "GDSC SMLM: Single-molecule localisation microscopy software for ImageJ." Wellcome Open Research 7 (September 29, 2022): 241. http://dx.doi.org/10.12688/wellcomeopenres.18327.1.
Анотація:
Single-molecule localisation microscopy (SMLM) uses software to extract super-resolved positions from microscope images of fluorescent molecules. These localisations can then be used to render super-resolution images or analysed to extract information about molecular behaviour. The GDSC SMLM software provides a set of tools for analysing SMLM data in a single cross-platform environment. The software identifies fluorescent molecules in raw microscope images and localises their positions using stages of spot detection, spot fitting and spot rejection. The resulting localisation data set can then be visualised, cropped and filtered. A suite of downstream analysis tools enable the user to perform single-particle tracking, cluster analysis and drift correction. In addition, GDSC SMLM also provides utility tools that enable modelling of EM-CCD and sCMOS cameras as well as point spread functions (PSFs) for data simulation. The software is written in Java and runs as a collection of plugins for the ImageJ software.
45
Schavkan, Alexander, Christian Gollwitzer, Raul Garcia-Diez, Michael Krumrey, Caterina Minelli, Dorota Bartczak, Susana Cuello-Nuñez, et al. "Number Concentration of Gold Nanoparticles in Suspension: SAXS and spICPMS as Traceable Methods Compared to Laboratory Methods." Nanomaterials 9, no. 4 (April 1, 2019): 502. http://dx.doi.org/10.3390/nano9040502.
Анотація:
The industrial exploitation of high value nanoparticles is in need of robust measurement methods to increase the control over product manufacturing and to implement quality assurance. InNanoPart, a European metrology project responded to these needs by developing methods for the measurement of particle size, concentration, agglomeration, surface chemistry and shell thickness. This paper illustrates the advancements this project produced for the traceable measurement of nanoparticle number concentration in liquids through small angle X-ray scattering (SAXS) and single particle inductively coupled plasma mass spectrometry (spICPMS). It also details the validation of a range of laboratory methods, including particle tracking analysis (PTA), dynamic light scattering (DLS), differential centrifugal sedimentation (DCS), ultraviolet visible spectroscopy (UV-vis) and electrospray-differential mobility analysis with a condensation particle counter (ES-DMA-CPC). We used a set of spherical gold nanoparticles with nominal diameters between 10 nm and 100 nm and discuss the results from the various techniques along with the associated uncertainty budgets.
46
Ostrovskii, Igor. "Resonant ultrasound spectroscopy of free vibrating oscillators for massive particles detection." Journal of the Acoustical Society of America 150, no. 4 (October 2021): A335. http://dx.doi.org/10.1121/10.0008492.
Анотація:
The half-inch diameter quartz oscillators are set for free vibrations. The crystal admittance (Y) versus frequency of ultrasound (F) is measured near fundamental harmonic of 9-10 MHz. The quartz crystallographic axes X, Y, interplain and rotated are oriented in space toward the sky locations of the Sun, center of the Milky Way galaxy or another star. It is demonstrated that Y(F) spectra are sensitive to an alignment of the crystallographic axes with space direction toward the stars. The two general results include: (1) changing Y(F) from a few to tens of percents under aligning crystallographic axes toward the stars and (2) occurring periodic minima in Y(F)-dependencies taken during some seconds with a tracking generator synchronized with a spectrum analyzer. The deepest minima in Y(F) appear when the azimuths of crystal axes coincide with those of the stars. These changes may be explained by electrically neutral massive particles (mp) emitted from a star and interacting with the crystals. The mass of a single mp-particle is estimated around ∼5 × 10−21 kg. RUS measurements correlate with the data from a crystal pendulum having a quartz bob and starts swinging when mp-particle hits it. RUS of free vibrating oscillators is perspective for detecting stellar massive particles.
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Pak, Alexander J., John M. A. Grime, Prabuddha Sengupta, Antony K. Chen, Aleksander E. P. Durumeric, Anand Srivastava, Mark Yeager, John A. G. Briggs, Jennifer Lippincott-Schwartz, and Gregory A. Voth. "Immature HIV-1 lattice assembly dynamics are regulated by scaffolding from nucleic acid and the plasma membrane." Proceedings of the National Academy of Sciences 114, no. 47 (November 7, 2017): E10056—E10065. http://dx.doi.org/10.1073/pnas.1706600114.
Анотація:
The packaging and budding of Gag polyprotein and viral RNA is a critical step in the HIV-1 life cycle. High-resolution structures of the Gag polyprotein have revealed that the capsid (CA) and spacer peptide 1 (SP1) domains contain important interfaces for Gag self-assembly. However, the molecular details of the multimerization process, especially in the presence of RNA and the cell membrane, have remained unclear. In this work, we investigate the mechanisms that work in concert between the polyproteins, RNA, and membrane to promote immature lattice growth. We develop a coarse-grained (CG) computational model that is derived from subnanometer resolution structural data. Our simulations recapitulate contiguous and hexameric lattice assembly driven only by weak anisotropic attractions at the helical CA–SP1 junction. Importantly, analysis from CG and single-particle tracking photoactivated localization (spt-PALM) trajectories indicates that viral RNA and the membrane are critical constituents that actively promote Gag multimerization through scaffolding, while overexpression of short competitor RNA can suppress assembly. We also find that the CA amino-terminal domain imparts intrinsic curvature to the Gag lattice. As a consequence, immature lattice growth appears to be coupled to the dynamics of spontaneous membrane deformation. Our findings elucidate a simple network of interactions that regulate the early stages of HIV-1 assembly and budding.
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Cao, Yangyang, Qizouhong He, Zengxing Qi, Yan Zhang, Liang Lu, Jingyuan Xue, Junling Li, and Ruili Li. "Dynamics and Endocytosis of Flot1 in Arabidopsis Require CPI1 Function." International Journal of Molecular Sciences 21, no. 5 (February 25, 2020): 1552. http://dx.doi.org/10.3390/ijms21051552.
Анотація:
Membrane microdomains are nano-scale domains (10–200 nm) enriched in sterols and sphingolipids. They have many important biological functions, including vesicle transport, endocytosis, and pathogen invasion. A previous study reported that the membrane microdomain-associated protein Flotillin1 (Flot1) was involved in plant development in Arabidopsis thaliana; however, whether sterols affect the plant immunity conveyed by Flot1 is unknown. Here, we showed that the root length in sterol-deficient cyclopropylsterol isomerase 1 (cpi1-1) mutants expressing Flot1 was significantly shorter than in control seedlings. The cotyledon epidermal cells in cpi1-1 mutants expressing Flot1 were smaller than in controls. Moreover, variable-angle total internal reflection fluorescence microscopy (VA-TIRFM) and single-particle tracking (SPT) analysis demonstrated that the long-distance Flot1-GFP movement was decreased significantly in cpi1-1 mutants compared with the control seedlings. Meanwhile, the value of the diffusion coefficient Ĝ was dramatically decreased in cpi1-1 mutants after flagelin22 (flg22) treatment compared with the control seedlings, indicating that sterols affect the lateral mobility of Flot1-GFP within the plasma membrane. Importantly, using confocal microscopy, we determined that the endocytosis of Flot1-GFP was decreased in cpi1-1 mutants, which was confirmed by fluorescence cross spectroscopy (FCS) analysis. Hence, these results demonstrate that sterol composition plays a critical role in the plant defense responses of Flot1.
49
Butail, Sachit, Nicholas Manoukis, Moussa Diallo, José M. Ribeiro, Tovi Lehmann, and Derek A. Paley. "Reconstructing the flight kinematics of swarming and mating in wild mosquitoes." Journal of The Royal Society Interface 9, no. 75 (May 23, 2012): 2624–38. http://dx.doi.org/10.1098/rsif.2012.0150.
Анотація:
We describe a novel tracking system for reconstructing three-dimensional tracks of individual mosquitoes in wild swarms and present the results of validating the system by filming swarms and mating events of the malaria mosquito Anopheles gambiae in Mali. The tracking system is designed to address noisy, low frame-rate (25 frames per second) video streams from a stereo camera system. Because flying A. gambiae move at 1–4 m s −1 , they appear as faded streaks in the images or sometimes do not appear at all. We provide an adaptive algorithm to search for missing streaks and a likelihood function that uses streak endpoints to extract velocity information. A modified multi-hypothesis tracker probabilistically addresses occlusions and a particle filter estimates the trajectories. The output of the tracking algorithm is a set of track segments with an average length of 0.6–1 s. The segments are verified and combined under human supervision to create individual tracks up to the duration of the video (90 s). We evaluate tracking performance using an established metric for multi-target tracking and validate the accuracy using independent stereo measurements of a single swarm. Three-dimensional reconstructions of A. gambiae swarming and mating events are presented.
50
Böyük, Mustafa, Yakup Eroğlu, Günyaz Ablay, and Kutay İçöz. "Feedback controller designs for an electromagnetic micromanipulator." Proceedings of the Institution of Mechanical Engineers, Part I: Journal of Systems and Control Engineering 234, no. 6 (September 9, 2019): 759–72. http://dx.doi.org/10.1177/0959651819871783.
Анотація:
Magnetic micromanipulators are capable of generating wide range of magnetic forces to manipulate magnetic microparticles for biomedical applications. In this study, a multipole magnetic micromanipulator system including electromagnets, driver circuitry and control unit is designed, modeled and implemented. The micromanipulator can produce a broad range of magnetic forces up to 25 pN on a single magnetic microparticle (1–10 µm diameter) that is 5 mm away from the electromagnet core tip. Both linear and nonlinear controllers are designed and implemented, and the proposed nonlinear controller produces smooth control currents to assure closed-loop stability of the system with 1 s non-overshoot transient response and zero steady-state tracking error. The maximum output current of the driver circuitry is set to 1 A. The single particle at the center is moved at a speed of 5 mm/s. The fully automatic system can be utilized in applications related to single cell or microparticle manipulations.