Single-Molecule Identification in Flowing Sample Streams by Fluorescence Burst Size and Intraburst Fluorescence Decay Rate

1998 ◽  
Vol 70 (7) ◽  
pp. 1444-1451 ◽  
Author(s):  
Alan Van Orden ◽  
Nicholas P. Machara ◽  
Peter M. Goodwin ◽  
Richard A. Keller
Keyword(s):  
Decay Rate ◽  
Single Molecule ◽  
Burst Size ◽  
Fluorescence Decay ◽  
Flowing Sample

scholarly journals Single-molecule tracking of tau reveals fast kiss-and-hop interaction with microtubules in living neurons

2014 ◽  
Vol 25 (22) ◽  
pp. 3541-3551 ◽  
Author(s):  
Dennis Janning ◽  
Maxim Igaev ◽  
Frederik Sündermann ◽  
Jörg Brühmann ◽  
Oliver Beutel ◽  
...  
Keyword(s):  
Single Molecule ◽  
Dwell Time ◽  
Quantitative Imaging ◽  
Fluorescence Decay ◽  
Microtubule Dynamics ◽  
Microtubule Assembly ◽  
Apparent Paradox ◽  
Single Molecule Tracking ◽  
Dependent Transport ◽  
Living Neurons

The microtubule-associated phosphoprotein tau regulates microtubule dynamics and is involved in neurodegenerative diseases collectively called tauopathies. It is generally believed that the vast majority of tau molecules decorate axonal microtubules, thereby stabilizing them. However, it is an open question how tau can regulate microtubule dynamics without impeding microtubule-dependent transport and how tau is also available for interactions other than those with microtubules. Here we address this apparent paradox by fast single-molecule tracking of tau in living neurons and Monte Carlo simulations of tau dynamics. We find that tau dwells on a single microtubule for an unexpectedly short time of ∼40 ms before it hops to the next. This dwell time is 100-fold shorter than previously reported by ensemble measurements. Furthermore, we observed by quantitative imaging using fluorescence decay after photoactivation recordings of photoactivatable GFP–tagged tubulin that, despite this rapid dynamics, tau is capable of regulating the tubulin–microtubule balance. This indicates that tau's dwell time on microtubules is sufficiently long to influence the lifetime of a tubulin subunit in a GTP cap. Our data imply a novel kiss-and-hop mechanism by which tau promotes neuronal microtubule assembly. The rapid kiss-and-hop interaction explains why tau, although binding to microtubules, does not interfere with axonal transport.

scholarly journals Single-molecule imaging reveals the interplay between transcription factors, nucleosomes, and transcriptional bursting

2018 ◽  
Author(s):  
Benjamin T. Donovan ◽  
Anh Huynh ◽  
David A. Ball ◽  
Michael G. Poirier ◽  
Daniel R. Larson ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Transcription Factors ◽  
Single Molecule ◽  
Target Genes ◽  
Burst Size ◽  
Yeast Cells ◽  
Single Molecule Imaging ◽  
Transcriptional Bursting

SummaryTranscription factors show rapid and reversible binding to chromatin in living cells, and transcription occurs in sporadic bursts, but how these phenomena are related is unknown. Using a combination of in vitro and in vivo single-molecule imaging approaches, we directly correlated binding of the transcription factor Gal4 with the transcriptional bursting kinetics of the Gal4 target genes GAL3 and GAL10 in living yeast cells. We find that Gal4 dwell times sets the transcriptional burst size. Gal4 dwell time depends on the affinity of the binding site and is reduced by orders of magnitude by nucleosomes. Using a novel imaging platform, we simultaneously tracked transcription factor binding and transcription at one locus, revealing the timing and correlation between Gal4 binding and transcription. Collectively, our data support a model where multiple polymerases initiate during a burst as long as the transcription factor is bound to DNA, and a burst terminates upon transcription factor dissociation.

Single-Molecule Detection in the Near-IR Using Continuous-Wave Diode Laser Excitation with an Avalanche Photon Detector

1998 ◽  
Vol 52 (1) ◽  
pp. 1-6 ◽  
Author(s):  
Steven A. Soper ◽  
Benjamin L. Legendre
Keyword(s):  
Diode Laser ◽  
Single Molecule ◽  
Continuous Wave ◽  
Single Photon ◽  
Single Molecules ◽  
Single Molecule Detection ◽  
Semiconductor Diode ◽  
Semiconductor Diode Laser ◽  
Near Ir ◽  
Flowing Sample

While single-molecule detection in flowing sample streams has been reported by a number of groups, the instrumentation can be somewhat prohibitive for many applications due to the complexity and extensive expertise required to operate such a device. In this paper we report on the construction of a single-molecule detection device that is rugged, compact, inexpensive, and easily operated by individuals not well trained in optics and laser operations. The single-molecule detection apparatus consists of a semiconductor diode laser operating in a continuous-wave (CW) mode and a single photon avalanche diode transducer for converting the detected photons into transistor–transistor logic (TTL) pulses for displaying the data. In addition, the sampling volume is produced by a single-component lens, to create a volume on the order of 1 pL, allowing the sampling of microliter volumes of material on reasonable time scales. The device is targeted for operation in the near-IR region (700–1000 nm), where matrix interferences are minimal. Our data will demonstrate the detection of single molecules for the near-IR dyes IR-132 and IR-125, in methanol solvents in flowing sample streams at sampling rates of 100–250 samples/s. Detection efficiencies for the investigated near-IR dyes were found to be 98% for IR-132 and 50% for IR-125. Previous attempts in our laboratory to detect single molecules of IR-125 using time-gated detection were unsuccessful because of the short upper-state lifetime of this fluorophore (τf = 472 ps).

scholarly journals A Test for Bottlenecks in the Vibrational Relaxation of CH3Cl and CH3Br by Ar

1988 ◽  
Vol 9 (1-3) ◽  
pp. 47-62 ◽  
Author(s):  
Kenneth M. Beck ◽  
Robert J. Gordon
Keyword(s):  
Decay Rate ◽  
Relaxation Rate ◽  
Vibrational Relaxation ◽  
Energy Levels ◽  
Fluorescence Decay ◽  
Rotational Relaxation ◽  
Translational Energy ◽  
Time Resolved ◽  
Level Model ◽  
Relaxing Gas

The method of time-resolved optoacoustics was used to measure the rate of vibrational relaxation of CH3Cl(ν6) and CH3Br(ν6) by Ar. The pressure pulses generated by the relaxing gas revealed that the rate of production of translational energy from ν6 = 1 is approximately twice the decay rate of IR fluorescence from ν3 = 1. No evidence was found for a previously proposed bottleneck in rotational relaxation, which would have resulted in an acoustic relaxation rate slower than the fluorescence decay. The faster rates observed here can be explained qualitatively by a rapid energy release from energy levels above ν3 which precedes the IR fluorescence. A simple three-level model, however, is unable to explain our observations quantitatively.

scholarly journals Presence of a carboxy-terminal pseudorepeat and disease-like pseudohyperphosphorylation critically influence tau’s interaction with microtubules in axon-like processes

2016 ◽  
Vol 27 (22) ◽  
pp. 3537-3549 ◽  
Author(s):  
Benedikt Niewidok ◽  
Maxim Igaev ◽  
Frederik Sündermann ◽  
Dennis Janning ◽  
Lidia Bakota ◽  
...  
Keyword(s):  
Single Molecule ◽  
Cell Biology ◽  
Major Change ◽  
Bioinformatic Analysis ◽  
Fluorescence Decay ◽  
Dissociation Rate ◽  
Tau Hyperphosphorylation ◽  
A Minor ◽  
Carboxy Terminal

A current challenge of cell biology is to investigate molecular interactions in subcellular compartments of living cells to overcome the artificial character of in vitro studies. To dissect the interaction of the neuronal microtubule (MT)-associated protein tau with MTs in axon-like processes, we used a refined fluorescence decay after photoactivation approach and single-molecule tracking. We found that isoform variation had only a minor influence on the tau–MT interaction, whereas the presence of a C-terminal pseudorepeat region (PRR) greatly increased MT binding by a greater-than-sixfold reduction of the dissociation rate. Bioinformatic analysis revealed that the PRR contained a highly conserved motif of 18 amino acids. Disease-associated tau mutations in the PRR (K369I, G389R) did not influence apparent MT binding but increased its dynamicity. Simulation of disease-like tau hyperphosphorylation dramatically diminished the tau–MT interaction by a greater-than-fivefold decrease of the association rate with no major change in the dissociation rate. Apparent binding of tau to MTs was similar in axons and dendrites but more sensitive to increased phosphorylation in axons. Our data indicate that under the conditions of high MT density that prevail in the axon, tau’s MT binding and localization are crucially affected by the presence of the PRR and tau hyperphosphorylation.

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