scholarly journals Single-Molecule Fluorescence and in Vivo Optical Traps: How Multiple Dyneins and Kinesins Interact

2014 ◽  
Vol 114 (6) ◽  
pp. 3335-3352 ◽  
Author(s):  
Benjamin H. Blehm ◽  
Paul R. Selvin
Keyword(s):  
Single Molecule ◽  
Optical Traps ◽  
Single Molecule Fluorescence

scholarly journals Bio-Molecular Applications of Recent Developments in Optical Tweezers

Biomolecules ◽  
2019 ◽  
Vol 9 (1) ◽  
pp. 23 ◽  
Author(s):  
Dhawal Choudhary ◽  
Alessandro Mossa ◽  
Milind Jadhav ◽  
Ciro Cecconi
Keyword(s):  
Photonic Crystal ◽  
Optical Tweezers ◽  
Single Molecule ◽  
Continuous Wave ◽  
Imaging Techniques ◽  
Resolving Power ◽  
Optical Traps ◽  
Laser Source ◽  
One Dimensional

In the past three decades, the ability to optically manipulate biomolecules has spurred a new era of medical and biophysical research. Optical tweezers (OT) have enabled experimenters to trap, sort, and probe cells, as well as discern the structural dynamics of proteins and nucleic acids at single molecule level. The steady improvement in OT’s resolving power has progressively pushed the envelope of their applications; there are, however, some inherent limitations that are prompting researchers to look for alternatives to the conventional techniques. To begin with, OT are restricted by their one-dimensional approach, which makes it difficult to conjure an exhaustive three-dimensional picture of biological systems. The high-intensity trapping laser can damage biological samples, a fact that restricts the feasibility of in vivo applications. Finally, direct manipulation of biological matter at nanometer scale remains a significant challenge for conventional OT. A significant amount of literature has been dedicated in the last 10 years to address the aforementioned shortcomings. Innovations in laser technology and advances in various other spheres of applied physics have been capitalized upon to evolve the next generation OT systems. In this review, we elucidate a few of these developments, with particular focus on their biological applications. The manipulation of nanoscopic objects has been achieved by means of plasmonic optical tweezers (POT), which utilize localized surface plasmons to generate optical traps with enhanced trapping potential, and photonic crystal optical tweezers (PhC OT), which attain the same goal by employing different photonic crystal geometries. Femtosecond optical tweezers (fs OT), constructed by replacing the continuous wave (cw) laser source with a femtosecond laser, promise to greatly reduce the damage to living samples. Finally, one way to transcend the one-dimensional nature of the data gained by OT is to couple them to the other large family of single molecule tools, i.e., fluorescence-based imaging techniques. We discuss the distinct advantages of the aforementioned techniques as well as the alternative experimental perspective they provide in comparison to conventional OT.

scholarly journals Stable end-sealed DNA as robust nano-rulers for in vivo single-molecule fluorescence

2016 ◽  
Vol 7 (7) ◽  
pp. 4418-4422 ◽  
Author(s):  
A. Plochowietz ◽  
A. H. El-Sagheer ◽  
T. Brown ◽  
A. N. Kapanidis
Keyword(s):  
Single Molecule ◽  
Single Molecule Fluorescence

Protected DNA standards with chemically linked ends were synthesized as robust in vivo nano-rulers for smFRET studies.

scholarly journals Mechanically switching single-molecule fluorescence of GFP by unfolding and refolding

2017 ◽  
Vol 114 (42) ◽  
pp. 11052-11056 ◽  
Author(s):  
Ziad Ganim ◽  
Matthias Rief
Keyword(s):  
Optical Tweezers ◽  
Single Molecule ◽  
Optical Sensors ◽  
Structural Integrity ◽  
Fluorescent Protein ◽  
Single Molecule Fluorescence ◽  
Extended States ◽  
Green Fluorescent ◽  
Ensemble Experiments

Green fluorescent protein (GFP) variants are widely used as genetically encoded fluorescent fusion tags, and there is an increasing interest in engineering their structure to develop in vivo optical sensors, such as for optogenetics and force transduction. Ensemble experiments have shown that the fluorescence of GFP is quenched upon denaturation. Here we study the dependence of fluorescence on protein structure by driving single molecules of GFP into different conformational states with optical tweezers and simultaneously probing the chromophore with fluorescence. Our results show that fluorescence is lost during the earliest events in unfolding, 3.5 ms before secondary structure is disrupted. No fluorescence is observed from the unfolding intermediates or the ensemble of compact and extended states populated during refolding. We further demonstrate that GFP can be mechanically switched between emissive and dark states. These data definitively establish that complete structural integrity is necessary to observe single-molecule fluorescence of GFP.

Nucleic acid amplification-integrated single-molecule fluorescence imaging for in vitro and in vivo biosensing

2021 ◽  
Author(s):  
Fei Ma ◽  
Chen-Chen Li ◽  
Chun-Yang Zhang
Keyword(s):  
Nucleic Acid ◽  
Fluorescence Imaging ◽  
Single Molecule ◽  
High Sensitivity ◽  
Nucleic Acid Amplification ◽  
Single Molecule Fluorescence

Single-molecule fluorescence imaging is among the most advanced analytical technologies and has been widely adopted for biosensing due to its distinct advantages of simplicity, rapidity, high sensitivity, low sample consumption,...

scholarly journals Inferring quantity and qualities of superimposed reaction rates in single molecule survival time distributions

2019 ◽  
Author(s):  
Matthias Reisser ◽  
Johannes Hettich ◽  
Timo Kuhn ◽  
J. Christof M. Gebhardt
Keyword(s):  
Survival Time ◽  
Single Molecule ◽  
Rate Constants ◽  
Reaction Rates ◽  
Decay Rates ◽  
Single Molecule Fluorescence ◽  
Inverse Laplace Transformation ◽  
Dense Grid ◽  
Reaction Processes

Actions of molecular species, for example binding of transcription factors to chromatin, are intrinsically stochastic and may comprise several mutually exclusive pathways. Inverse Laplace transformation in principle resolves the rate constants and frequencies of superimposed reaction processes, however current approaches are challenged by single molecule fluorescence time series prone to photobleaching. Here, we present a genuine rate identification method (GRID) that infers the quantity, rates and frequencies of dissociation processes from single molecule fluorescence survival time distributions using a dense grid of possible decay rates. In particular, GRID is able to resolve broad clusters of rate constants not accessible to common models of one to three exponential decay rates. We validate GRID by simulations and apply it to the problem of in-vivo TF-DNA dissociation, which recently gained interest due to novel single molecule imaging technologies. We consider dissociation of the transcription factor CDX2 from chromatin. GRID resolves distinct, decay rates and identifies residence time classes overlooked by other methods. We confirm that such sparsely distributed decay rates are compatible with common models of TF sliding on DNA.

scholarly journals Single-molecule biophysics: at the interface of biology, physics and chemistry

2007 ◽  
Vol 5 (18) ◽  
pp. 15-45 ◽  
Author(s):  
Ashok A Deniz ◽  
Samrat Mukhopadhyay ◽  
Edward A Lemke
Keyword(s):  
Single Molecule ◽  
Biological Molecules ◽  
Single Molecule Fluorescence ◽  
Complex Behaviour ◽  
Single Molecule Biophysics ◽  
Single Molecule Studies ◽  
Dynamics And Function ◽  
And Function ◽  
Ensemble Experiments

Single-molecule methods have matured into powerful and popular tools to probe the complex behaviour of biological molecules, due to their unique abilities to probe molecular structure, dynamics and function, unhindered by the averaging inherent in ensemble experiments. This review presents an overview of the burgeoning field of single-molecule biophysics, discussing key highlights and selected examples from its genesis to our projections for its future. Following brief introductions to a few popular single-molecule fluorescence and manipulation methods, we discuss novel insights gained from single-molecule studies in key biological areas ranging from biological folding to experiments performed in vivo .

scholarly journals In Vivo Investigation of DNA Replication in Escherichia Coli using Single-Molecule Fluorescence Microscopy

2012 ◽  
Vol 102 (3) ◽  
pp. 284a
Author(s):  
Charl Moolman ◽  
Sriram Tiruvadi Krishnan ◽  
Serge Donkers ◽  
Rodrigo Reyes-Lamothe ◽  
David Sherratt ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Dna Replication ◽  
Fluorescence Microscopy ◽  
Single Molecule ◽  
Single Molecule Fluorescence ◽  
Single Molecule Fluorescence Microscopy
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