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

2020 ◽  
Author(s):  
Zhijie Chen ◽  
Alan Shaw ◽  
Hugh Wilson ◽  
Maxime Woringer ◽  
Xavier Darzacq ◽  
...  
Keyword(s):  
Alkaline Phosphatase ◽  
Single Molecule ◽  
Fluorescence Correlation Spectroscopy ◽  
Theoretical Models ◽  
Single Particle Tracking ◽  
Correlation Spectroscopy ◽  
Diffusion Behavior ◽  
Fluorescence Correlation ◽  
Apparent Diffusion ◽  
Diffusion Enhancement

ABSTRACTTheoretical 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. Experimental observations of catalysis-enhanced enzyme diffusion, to date, 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 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 (SPT), 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 using p-nitrophenyl phosphate (pNPP) as substrate. Combining comprehensive FCS controls, ABEL trap, surface-based single-molecule fluorescence, and Monte-Carlo simulations, we establish that pNPP-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 SPT and ABEL trap are more reliable means to investigate diffusion phenomena at the nanoscale.SIGNIFICANCE STATEMENTRecent experiments have suggested that the energy released by a chemical reaction can propel its enzyme catalyst (for example, alkaline phosphatase, ALP). However, this topic remains controversial, partially due to the indirect and ensemble nature of existing measurements. Here, we used recently developed single-molecule approaches to monitor directly the motions of individual proteins in aqueous solution and find that single ALP enzymes do not diffuse faster under catalysis. Instead, we demonstrate that interactions between the fluorescent dye and the enzyme’s substrate can produce the signature of apparent diffusion enhancement in fluorescence correlation spectroscopy (FCS), the standard ensemble assay currently used to study enzyme diffusion and indicate that single-molecule approaches provide a more robust means to investigate diffusion 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 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

Enhancing the generating and collecting efficiency of single particle upconverting luminescence at low power excitation

Nanophotonics ◽  
2020 ◽  
Vol 9 (7) ◽  
pp. 1993-2000 ◽  
Author(s):  
Chenshuo Ma ◽  
Chunyan Shan ◽  
Kevin Park ◽  
Aaron T. Mok ◽  
Paul J. Antonick ◽  
...  
Keyword(s):  
Single Molecule ◽  
Single Particle ◽  
Excitation Power ◽  
Single Particle Tracking ◽  
Rare Earth Ions ◽  
High Excitation ◽  
Single Molecule Level ◽  
Collecting Efficiency ◽  
High Luminescence Intensity

AbstractUpconverting luminescent nanoparticles are photostable, nonblinking, and low chemically toxic fluorophores that are emerging as promising fluorescent probes at the single molecule level. High luminescence intensity upconversion nanoparticles (UCNPs) have previously been achieved by doping with high amounts of rare-earth ions using high excitation power (>2.5 MW/cm2). However, such particles are inadequate for in vitro live-cell imaging and single-particle tracking, as high excitation power can cause photodamage. Here, we compared UCNP luminescence intensities with different dopant concentrations and presented more efficient (about seven times) UCNPs at low excitation power by increasing the concentrations of Yb3+ and Tm3+ dopants (NaYF4: 60% Yb3+, 8% Tm3+) and adding a core-shell structure.

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.

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