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

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.

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 Photoactivation of silicon rhodamines via a light-induced protonation

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Michelle S. Frei ◽  
Philipp Hoess ◽  
Marko Lampe ◽  
Bianca Nijmeijer ◽  
Moritz Kueblbeck ◽  
...  
Keyword(s):  
Single Molecule ◽  
Particle Tracking ◽  
Single Particle ◽  
Super Resolution ◽  
Spectroscopic Properties ◽  
Single Particle Tracking ◽  
Live Cell ◽  
Localization Microscopy ◽  
Super Resolution Microscopy ◽  
Single Molecule Localization Microscopy

Abstract Photoactivatable fluorophores are important for single-particle tracking and super-resolution microscopy. Here we present a photoactivatable fluorophore that forms a bright silicon rhodamine derivative through a light-dependent protonation. In contrast to other photoactivatable fluorophores, no caging groups are required, nor are there any undesired side-products released. Using this photoactivatable fluorophore, we create probes for HaloTag and actin for live-cell single-molecule localization microscopy and single-particle tracking experiments. The unusual mechanism of photoactivation and the fluorophore’s outstanding spectroscopic properties make it a powerful tool for live-cell super-resolution microscopy.

scholarly journals Single particle tracking-based reaction progress kinetic analysis reveals a series of molecular mechanisms of cetuximab-induced EGFR processes in a single living cell

2017 ◽  
Vol 8 (7) ◽  
pp. 4823-4832 ◽  
Author(s):  
Do-Hyeon Kim ◽  
Dong-Kyun Kim ◽  
Kai Zhou ◽  
Soyeon Park ◽  
Yonghoon Kwon ◽  
...  
Keyword(s):  
Kinetic Analysis ◽  
Single Molecule ◽  
Particle Tracking ◽  
Single Particle ◽  
Living Cell ◽  
Molecular Mechanisms ◽  
Single Particle Tracking ◽  
Reaction Progress ◽  
Single Living ◽  
Reaction Progress Kinetic Analysis

Reaction progress kinetic analysis utilizing single molecule trajectories revealed the comprehensive molecular mechanisms of cetuximab induced EGFR endocytosis.

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