scholarly journals Spot-On: robust model-based analysis of single-particle tracking experiments

2017 ◽  
Author(s):  
Anders S Hansen ◽  
Maxime Woringer ◽  
Jonathan B Grimm ◽  
Luke D Lavis ◽  
Robert Tjian ◽  
...  
Keyword(s):  
Single Molecule ◽  
Particle Tracking ◽  
Single Particle ◽  
Motion Blur ◽  
Single Particle Tracking ◽  
Modeling Framework ◽  
Diffusion Constants ◽  
Diffusion Dynamics ◽  
Wide Range ◽  
And Diffusion

ABSTRACTSingle-particle tracking (SPT) has become an important method to bridge biochemistry and cell biology since it allows direct observation of protein binding and diffusion dynamics in live cells. However, accurately inferring information from SPT studies is challenging due to biases in both data analysis and experimental design. To address analysis bias, we introduce “Spot-On”, an intuitive web-interface. Spot-On implements a kinetic modeling framework that accounts for known biases, including molecules moving out-of-focus, and robustly infers diffusion constants and subpopulations from pooled single-molecule trajectories. To minimize inherent experimental biases, we implement and validate stroboscopic photo-activation SPT (spaSPT), which minimizes motion-blur bias and tracking errors. We validate Spot-On using experimentally realistic simulations and show that Spot-On outperforms other methods. We then apply Spot-On to spaSPT data from live mammalian cells spanning a wide range of nuclear dynamics and demonstrate that Spot-On consistently and robustly infers subpopulation fractions and diffusion constants.IMPACT STATEMENTSpot-On is an easy-to-use website that makes a rigorous and bias-corrected modeling framework for analysis of single-molecule tracking experiments available to all.

scholarly journals Robust model-based analysis of single-particle tracking experiments with Spot-On

eLife ◽  
2018 ◽  
Vol 7 ◽  
Author(s):  
Anders S Hansen ◽  
Maxime Woringer ◽  
Jonathan B Grimm ◽  
Luke D Lavis ◽  
Robert Tjian ◽  
...  
Keyword(s):  
Particle Tracking ◽  
Single Particle ◽  
Cell Biology ◽  
Motion Blur ◽  
Single Particle Tracking ◽  
Modeling Framework ◽  
Diffusion Constants ◽  
Diffusion Dynamics ◽  
Wide Range ◽  
And Diffusion

Single-particle tracking (SPT) has become an important method to bridge biochemistry and cell biology since it allows direct observation of protein binding and diffusion dynamics in live cells. However, accurately inferring information from SPT studies is challenging due to biases in both data analysis and experimental design. To address analysis bias, we introduce ‘Spot-On’, an intuitive web-interface. Spot-On implements a kinetic modeling framework that accounts for known biases, including molecules moving out-of-focus, and robustly infers diffusion constants and subpopulations from pooled single-molecule trajectories. To minimize inherent experimental biases, we implement and validate stroboscopic photo-activation SPT (spaSPT), which minimizes motion-blur bias and tracking errors. We validate Spot-On using experimentally realistic simulations and show that Spot-On outperforms other methods. We then apply Spot-On to spaSPT data from live mammalian cells spanning a wide range of nuclear dynamics and demonstrate that Spot-On consistently and robustly infers subpopulation fractions and diffusion constants.

scholarly journals Functional Characterization of an Electromagnetic Perceptive Protein

2020 ◽  
Author(s):  
Sunayana Mitra ◽  
Carlo Barnaba ◽  
Jens Schmidt ◽  
Galit Pelled ◽  
Assaf A. Gilad
Keyword(s):  
Particle Tracking ◽  
Single Particle ◽  
Total Internal Reflection ◽  
Magnetic Stimulation ◽  
New Technologies ◽  
Cellular Function ◽  
Single Particle Tracking ◽  
Internal Reflection ◽  
Total Internal Reflection Fluorescence ◽  
Diffusion Dynamics

AbstractMagnetoreception, the response to geomagnetic fields is a well described phenomenon in nature. However, it is likely that convergent evolution led to different mechanisms in different organisms. One intriguing example is the unique Electromagnetic Perceptive Gene (EPG) from the glass catfish Kryptopterus vitreolus, that can remotely control cellular function, upon magnetic stimulation in in-vitro and in animal models. Here, we report for the first time the cellular location and orientation of the EPG protein. We utilized a differential labelling technique in determining that the EPG protein is a membrane anchored protein with an N-terminal extracellular domain. The kinetics and diffusion dynamics of the EPG protein in response to magnetic stimulation was also elucidated using single particle imaging and tracking. Pulse chase labelling and Total Internal Reflection Fluorescence (TIRF) imaging revealed an increase in EPG kinetics post magnetic stimulation activation at a single particle level. Trajectory analysis show notably different EPG protein kinetics before and after magnetic stimulation in both 2 (free vs bound particle) and 3 state (free vs intermediate vs bound particle) tracking models. These data serve to provide additional information that support and understand the underlying biophysical mechanisms behind EPG activation by magnetic stimulation. In conclusion, our results provide evidence for the basis of magnetoreception in EPG protein that would aid in future studies that seek to understand this novel mechanism. This study is important for understanding the phenomenon of magnetoreception as well as developing new technologies for magnetogenetics – the utilization of electromagnetic fields to remotely control cellular function.Graphical TOCElucidation of magnetoreception in a fish derived Electromagnetic Perceptive Gene (EPG), using genetic tagging and single particle tracking with Total Internal Reflection Fluorescence (TIRF) suggests changes in kinetics of membranal motion upon stimulation by magnetic field.

scholarly journals Single-molecule diffusometry reveals no catalysis-induced diffusion enhancement of alkaline phosphatase as proposed by FCS experiments

2020 ◽  
Vol 117 (35) ◽  
pp. 21328-21335
Author(s):  
Zhijie Chen ◽  
Alan Shaw ◽  
Hugh Wilson ◽  
Maxime Woringer ◽  
Xavier Darzacq ◽  
...  
Keyword(s):  
Alkaline Phosphatase ◽  
Single Molecule ◽  
Particle Tracking ◽  
Single Particle ◽  
Theoretical Models ◽  
Single Particle Tracking ◽  
Correlation Spectroscopy ◽  
Single Molecule Level ◽  
Diffusion Phenomena ◽  
Diffusion Enhancement

Theoretical and experimental observations that catalysis enhances the diffusion of enzymes have generated exciting implications about nanoscale energy flow, molecular chemotaxis, and self-powered nanomachines. However, contradictory claims on the origin, magnitude, and consequence of this phenomenon continue to arise. To date, experimental observations of catalysis-enhanced enzyme diffusion have relied almost exclusively on fluorescence correlation spectroscopy (FCS), a technique that provides only indirect, ensemble-averaged measurements of diffusion behavior. Here, using an anti-Brownian electrokinetic (ABEL) trap and in-solution single-particle tracking, we show that catalysis does not increase the diffusion of alkaline phosphatase (ALP) at the single-molecule level, in sharp contrast to the ∼20% enhancement seen in parallel FCS experiments usingp-nitrophenyl phosphate (pNPP) as substrate. Combining comprehensive FCS controls, ABEL trap, surface-based single-molecule fluorescence, and Monte Carlo simulations, we establish thatpNPP-induced dye blinking at the ∼10-ms timescale is responsible for the apparent diffusion enhancement seen in FCS. Our observations urge a crucial revisit of various experimental findings and theoretical models––including those of our own––in the field, and indicate that in-solution single-particle tracking and ABEL trap are more reliable means to investigate diffusion phenomena at the nanoscale.

Correlation between the translational and rotational diffusion of rod-shaped nanocargo on a lipid membrane revealed by single-particle tracking

Nanoscale ◽  
2019 ◽  
Vol 11 (20) ◽  
pp. 10080-10087 ◽  
Author(s):  
Liangna He ◽  
Yiliang Li ◽  
Lin Wei ◽  
Zhongju Ye ◽  
Hua Liu ◽  
...  
Keyword(s):  
Particle Tracking ◽  
Single Particle ◽  
Theoretical Basis ◽  
Rational Design ◽  
Lipid Membrane ◽  
Rotational Diffusion ◽  
Single Particle Tracking ◽  
Cellular Translocation ◽  
Diffusion Dynamics ◽  
Translational And Rotational Diffusion

Revealing the diffusion dynamics of nanoparticles on a lipid membrane plays an important role in a better understanding of the cellular translocation process and provides a theoretical basis for the rational design of delivery cargo.

scholarly journals Real-Time 3D Single Particle Tracking: Towards Active Feedback Single Molecule Spectroscopy in Live Cells

Molecules ◽  
2019 ◽  
Vol 24 (15) ◽  
pp. 2826 ◽  
Author(s):  
Shangguo Hou ◽  
Courtney Johnson ◽  
Kevin Welsher
Keyword(s):  
Fluorescence Spectroscopy ◽  
Real Time ◽  
Temporal Resolution ◽  
Single Molecule ◽  
Particle Tracking ◽  
Single Particle ◽  
Single Particle Tracking ◽  
Single Molecule Fluorescence ◽  
Single Molecule Fluorescence Spectroscopy ◽  
Active Feedback

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.

scholarly journals A global sampler of single particle tracking solutions for single molecule microscopy

PLoS ONE ◽  
2019 ◽  
Vol 14 (10) ◽  
pp. e0221865
Author(s):  
Michael Hirsch ◽  
Richard Wareham ◽  
Ji W. Yoon ◽  
Daniel J. Rolfe ◽  
Laura C. Zanetti-Domingues ◽  
...  
Keyword(s):  
Single Molecule ◽  
Particle Tracking ◽  
Single Particle ◽  
Single Particle Tracking ◽  
Single Molecule Microscopy
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