Florian Rückerl, Martin Lenz, Timo Betz, John Manzi, Jean-Louis Martiel, Mahassine Safouane, Rajaa Paterski-Boujemaa, Laurent Blanchoin, Cécile Sykes
Actin is one of the main components of the architecture of cells. Actin filaments form different polymer networks with versatile mechanical properties that depend on their spatial organization and the presence of cross-linkers. Here, we investigate the mechanical properties of actin bundles in the absence of cross-linkers. Bundles are polymerized from the surface of mDia1-coated latex beads, and deformed by manipulating both ends through attached beads held by optical tweezers, allowing us to record the applied force. Bundle properties are strikingly different from the ones of a homogeneous isotropic beam. Successive compression and extension leads to a decrease in the buckling force that we attribute to the bundle remaining slightly curved after the first deformation. Furthermore, we find that the bundle is solid, and stiff to bending, along the long axis, whereas it has a liquid and viscous behavior in the transverse direction. Interpretation of the force curves using a Maxwell visco-elastic model allows us to extract the bundle mechanical parameters and confirms that the bundle is composed of weakly coupled filaments. At short times, the bundle behaves as an elastic material, whereas at long times, filaments flow in the longitudinal direction, leading to bundle restructuring. Deviations from the model reveal a complex adaptive rheological behavior of bundles. Indeed, when allowed to anneal between phases of compression and extension, the bundle reinforces. Moreover, we find that the characteristic visco-elastic time is inversely proportional to the compression speed. Actin bundles are therefore not simple force transmitters, but instead, complex mechano-transducers that adjust their mechanics to external stimulation. In cells, where actin bundles are mechanical sensors, this property could contribute to their adaptability.
DOI
Friday, September 29, 2017
Enhanced optical confinement of dielectric nanoparticles by two-photon resonance transition
Aungtinee Kittiravechote, Anwar Usman, Hiroshi Masuhara and Ian Liau
Despite a tremendous success in the optical manipulation of microscopic particles, it remains a challenge to manipulate nanoparticles especially as the polarizability of the particles is small. With a picosecond-pulsed near-infrared laser, we demonstrated recently that the confinement of dye-doped polystyrene nanobeads is significantly enhanced relative to bare nanobeads of the same dimension. We attributed the enhancement to an additional term of the refractive index, which results from two-photon resonance between the dopant and the optical field. The optical confinement is profoundly enhanced as the half-wavelength of the laser falls either on the red side, or slightly away from the blue side, of the absorption band of the dopant. In contrast, the ability to confine the nanobeads is significantly diminished as the half-wavelength of the laser locates either at the peak, or on the blue side, of the absorption band. We suggest that the dispersively shaped polarizability of the dopant near the resonance is responsible to the distinctive spectral dependence of the optical confinement of nanobeads. This work advances our understanding of the underlying mechanism of the enhanced optical confinement of doped nanoparticles with a near-infrared pulsed laser, and might facilitate future research that benefits from effective sorting of selected nanoparticles beyond the limitations of previous approaches.
DOI
Despite a tremendous success in the optical manipulation of microscopic particles, it remains a challenge to manipulate nanoparticles especially as the polarizability of the particles is small. With a picosecond-pulsed near-infrared laser, we demonstrated recently that the confinement of dye-doped polystyrene nanobeads is significantly enhanced relative to bare nanobeads of the same dimension. We attributed the enhancement to an additional term of the refractive index, which results from two-photon resonance between the dopant and the optical field. The optical confinement is profoundly enhanced as the half-wavelength of the laser falls either on the red side, or slightly away from the blue side, of the absorption band of the dopant. In contrast, the ability to confine the nanobeads is significantly diminished as the half-wavelength of the laser locates either at the peak, or on the blue side, of the absorption band. We suggest that the dispersively shaped polarizability of the dopant near the resonance is responsible to the distinctive spectral dependence of the optical confinement of nanobeads. This work advances our understanding of the underlying mechanism of the enhanced optical confinement of doped nanoparticles with a near-infrared pulsed laser, and might facilitate future research that benefits from effective sorting of selected nanoparticles beyond the limitations of previous approaches.
DOI
Probing Photothermal Effects on Optically Trapped Gold Nanorods by Simultaneous Plasmon Spectroscopy and Brownian Dynamics Analysis
Daniel Andrén, Lei Shao, Nils Odebo Länk, Srdjan S. Aćimović, Peter Johansson, and Mikael Käll
Plasmonic gold nanorods are prime candidates for a variety of biomedical, spectroscopy, data storage, and sensing applications. It was recently shown that gold nanorods optically trapped by a focused circularly polarized laser beam can function as extremely efficient nanoscopic rotary motors. The system holds promise for applications ranging from nanofluidic flow control and nanorobotics to biomolecular actuation and analysis. However, to fully exploit this potential, one needs to be able to control and understand heating effects associated with laser trapping. We investigated photothermal heating of individual rotating gold nanorods by simultaneously probing their localized surface plasmon resonance spectrum and rotational Brownian dynamics over extended periods of time. The data reveal an extremely slow nanoparticle reshaping process, involving migration of the order of a few hundred atoms per minute, for moderate laser powers and a trapping wavelength close to plasmon resonance. The plasmon spectroscopy and Brownian analysis allows for separate temperature estimates based on the refractive index and the viscosity of the water surrounding a trapped nanorod. We show that both measurements yield similar effective temperatures, which correspond to the actual temperature at a distance of the order 10–15 nm from the particle surface. Our results shed light on photothermal processes on the nanoscale and will be useful in evaluating the applicability and performance of nanorod motors and optically heated nanoparticles for a variety of applications.
DOI
Plasmonic gold nanorods are prime candidates for a variety of biomedical, spectroscopy, data storage, and sensing applications. It was recently shown that gold nanorods optically trapped by a focused circularly polarized laser beam can function as extremely efficient nanoscopic rotary motors. The system holds promise for applications ranging from nanofluidic flow control and nanorobotics to biomolecular actuation and analysis. However, to fully exploit this potential, one needs to be able to control and understand heating effects associated with laser trapping. We investigated photothermal heating of individual rotating gold nanorods by simultaneously probing their localized surface plasmon resonance spectrum and rotational Brownian dynamics over extended periods of time. The data reveal an extremely slow nanoparticle reshaping process, involving migration of the order of a few hundred atoms per minute, for moderate laser powers and a trapping wavelength close to plasmon resonance. The plasmon spectroscopy and Brownian analysis allows for separate temperature estimates based on the refractive index and the viscosity of the water surrounding a trapped nanorod. We show that both measurements yield similar effective temperatures, which correspond to the actual temperature at a distance of the order 10–15 nm from the particle surface. Our results shed light on photothermal processes on the nanoscale and will be useful in evaluating the applicability and performance of nanorod motors and optically heated nanoparticles for a variety of applications.
DOI
β1-Integrin-Mediated Adhesion Is Lipid-Bilayer Dependent
Seoyoung Son, George J. Moroney, Peter J.Butler
Integrin-mediated adhesion is a central feature of cellular adhesion, locomotion, and endothelial cell mechanobiology. Although integrins are known to be transmembrane proteins, little is known about the role of membrane biophysics and dynamics in integrin adhesion. We treated human aortic endothelial cells with exogenous amphiphiles, shown previously in model membranes, and computationally, to affect bilayer thickness and lipid phase separation, and subsequently measured single-integrin-molecule adhesion kinetics using an optical trap, and diffusion using fluorescence correlation spectroscopy. Benzyl alcohol (BA) partitions to liquid-disordered (Ld) domains, thins them, and causes the greatest increase in hydrophobic mismatch between liquid-ordered (Lo) and Ld domains among the three amphiphiles, leading to domain separation. In human aortic endothelial cells, BA increased β1-integrin-Arg-Gly-Asp-peptide affinity by 18% with a transition from single to double valency, consistent with a doubling of the molecular brightness of mCherry-tagged β1-integrins measured using fluorescence correlation spectroscopy. Accordingly, BA caused an increase in the size of focal-adhesion-kinase/paxillin-positive peripheral adhesions and reduced migration speeds as measured using wound-healing assays. Vitamin E, which thickens Lo domains and disperses them by lowering edge energy on domain boundaries, left integrin affinity unchanged but reduced binding probability, leading to smaller focal adhesions and equivalent migration speed relative to untreated cells. Vitamin E reversed the BA-induced decrease in migration speed. Triton X-100 also thickens Lo domains, but partitions to both lipid phases and left unchanged binding kinetics, focal adhesion sizes, and migration speed. These results demonstrate that only the amphiphile that thinned Ld lipid domains increased β1-integrin-Arg-Gly-Asp-peptide affinity and valency, thus implicating Ld domains in modulation of integrin adhesion, nascent adhesion formation, and cell migration.
DOI
Integrin-mediated adhesion is a central feature of cellular adhesion, locomotion, and endothelial cell mechanobiology. Although integrins are known to be transmembrane proteins, little is known about the role of membrane biophysics and dynamics in integrin adhesion. We treated human aortic endothelial cells with exogenous amphiphiles, shown previously in model membranes, and computationally, to affect bilayer thickness and lipid phase separation, and subsequently measured single-integrin-molecule adhesion kinetics using an optical trap, and diffusion using fluorescence correlation spectroscopy. Benzyl alcohol (BA) partitions to liquid-disordered (Ld) domains, thins them, and causes the greatest increase in hydrophobic mismatch between liquid-ordered (Lo) and Ld domains among the three amphiphiles, leading to domain separation. In human aortic endothelial cells, BA increased β1-integrin-Arg-Gly-Asp-peptide affinity by 18% with a transition from single to double valency, consistent with a doubling of the molecular brightness of mCherry-tagged β1-integrins measured using fluorescence correlation spectroscopy. Accordingly, BA caused an increase in the size of focal-adhesion-kinase/paxillin-positive peripheral adhesions and reduced migration speeds as measured using wound-healing assays. Vitamin E, which thickens Lo domains and disperses them by lowering edge energy on domain boundaries, left integrin affinity unchanged but reduced binding probability, leading to smaller focal adhesions and equivalent migration speed relative to untreated cells. Vitamin E reversed the BA-induced decrease in migration speed. Triton X-100 also thickens Lo domains, but partitions to both lipid phases and left unchanged binding kinetics, focal adhesion sizes, and migration speed. These results demonstrate that only the amphiphile that thinned Ld lipid domains increased β1-integrin-Arg-Gly-Asp-peptide affinity and valency, thus implicating Ld domains in modulation of integrin adhesion, nascent adhesion formation, and cell migration.
DOI
Enhancing Upconversion Fluorescence with a Natural Bio-microlens
Yuchao Li, Xiaoshuai Liu, Xianguang Yang, Hongxiang Lei, Yao Zhang, and Baojun Li
Upconversion fluorescence has triggered extensive efforts in the past decade because of its superior physicochemical features and great potential in biomedical and biophotonic studies. However, practical applications of upconversion fluorescence are often hindered by its relatively low luminescence efficiency (<1%). Here, we employ a living yeast or human cell as a natural bio-microlens to enhance the upconversion fluorescence. The natural bio-microlens, which was stably trapped on a fiber probe, could concentrate the excitation light into a subwavelength region so that the upconversion fluorescence of core–shell NaYF4:Yb3+/Tm3+ nanoparticles was enhanced by 2 orders of magnitude. As a benefit of the fluorescence enhancement, single-cell imaging and real-time detection of the labeled pathogenic bacteria, such as Escherichia coli and Staphylococcus aureus, were successfully achieved in the dark fields. This biocompatible, sensitive, and miniature approach could provide a promising powerful tool for biological imaging, biophotonic sensing, and single-cell analysis.
DOI
Upconversion fluorescence has triggered extensive efforts in the past decade because of its superior physicochemical features and great potential in biomedical and biophotonic studies. However, practical applications of upconversion fluorescence are often hindered by its relatively low luminescence efficiency (<1%). Here, we employ a living yeast or human cell as a natural bio-microlens to enhance the upconversion fluorescence. The natural bio-microlens, which was stably trapped on a fiber probe, could concentrate the excitation light into a subwavelength region so that the upconversion fluorescence of core–shell NaYF4:Yb3+/Tm3+ nanoparticles was enhanced by 2 orders of magnitude. As a benefit of the fluorescence enhancement, single-cell imaging and real-time detection of the labeled pathogenic bacteria, such as Escherichia coli and Staphylococcus aureus, were successfully achieved in the dark fields. This biocompatible, sensitive, and miniature approach could provide a promising powerful tool for biological imaging, biophotonic sensing, and single-cell analysis.
DOI
Optical Force Enhancement Using an Imaginary Vector Potential for Photons
Lana Descheemaeker, Vincent Ginis, Sophie Viaene, and Philippe Tassin
The enhancement of optical forces has enabled a variety of technological applications that rely on the optical control of small objects and devices. Unfortunately, optical forces are still too small for the convenient actuation of integrated switches and waveguide couplers. Here we show how the optical gradient force can be enhanced by an order of magnitude by making use of gauge materials inside two evanescently coupled waveguides. To this end, the gauge materials inside the cores should emulate imaginary vector potentials for photons pointing perpendicularly to the waveguide plane. Depending on the relative orientation of the vector potentials in neighboring waveguides, i.e., pointing away from or towards each other, the conventional attractive force due to an even mode profile may be enhanced, suppressed, or may even become repulsive. This and other new features indicate that the implementation of complex-valued vector potentials with non-Hermitian waveguide cores may further enhance our control over mode profiles and the associated optical forces.
DOI
The enhancement of optical forces has enabled a variety of technological applications that rely on the optical control of small objects and devices. Unfortunately, optical forces are still too small for the convenient actuation of integrated switches and waveguide couplers. Here we show how the optical gradient force can be enhanced by an order of magnitude by making use of gauge materials inside two evanescently coupled waveguides. To this end, the gauge materials inside the cores should emulate imaginary vector potentials for photons pointing perpendicularly to the waveguide plane. Depending on the relative orientation of the vector potentials in neighboring waveguides, i.e., pointing away from or towards each other, the conventional attractive force due to an even mode profile may be enhanced, suppressed, or may even become repulsive. This and other new features indicate that the implementation of complex-valued vector potentials with non-Hermitian waveguide cores may further enhance our control over mode profiles and the associated optical forces.
DOI
Thursday, September 21, 2017
Mitotic tethers connect sister chromosomes and transmit “cross-polar” force during anaphase A of mitosis in PtK2 cells
Matthew Ono, Daryl Preece, Michelle L. Duquette, Arthur Forer, and Michael W. Berns
Originally described in crane-fly spermatocytes, tethers physically link and transmit force between the ends of separating chromosomes. Optical tweezers and laser scissors were used to sever the tether between chromosomes, create chromosome fragments attached to the tether which move toward the opposite pole, and to trap the tethered fragments. Laser microsurgery in the intracellular space between separating telomeres reduced chromosome strain in half of tested chromosome pairs. When the telomere-containing region was severed from the rest of the chromosome body, the resultant fragment either traveled towards the proper pole (poleward), towards the sister pole (cross-polar), or movement ceased. Fragment travel towards the sister pole varied in distance and always ceased following a cut between telomeres, indicating the tether is responsible for transferring a cross-polar force to the fragment. Optical trapping of cross-polar traveling fragments places an upper boundary on the tethering force of ~1.5 pN.
DOI
Originally described in crane-fly spermatocytes, tethers physically link and transmit force between the ends of separating chromosomes. Optical tweezers and laser scissors were used to sever the tether between chromosomes, create chromosome fragments attached to the tether which move toward the opposite pole, and to trap the tethered fragments. Laser microsurgery in the intracellular space between separating telomeres reduced chromosome strain in half of tested chromosome pairs. When the telomere-containing region was severed from the rest of the chromosome body, the resultant fragment either traveled towards the proper pole (poleward), towards the sister pole (cross-polar), or movement ceased. Fragment travel towards the sister pole varied in distance and always ceased following a cut between telomeres, indicating the tether is responsible for transferring a cross-polar force to the fragment. Optical trapping of cross-polar traveling fragments places an upper boundary on the tethering force of ~1.5 pN.
DOI
All-dielectric structure for trapping nanoparticles via light funneling and nanofocusing
Amir M. Jazayeri and Khashayar Mehrany
We propose a dielectric structure which focuses laser light well beyond the diffraction limit and thus considerably enhances the exerted optical trapping force upon dielectric nanoparticles. Although the structure supports a Fabry–Perot resonance, it actually acts as a nanoantenna in that the role of the resonance is to funnel the laser light into the structure. In comparison with the lens illuminating the structure, the proposed structure offers roughly a 10,000-fold enhancement in the trapping force—part of this enhancement comes from an 80-fold enhancement in the field intensity, whereas the remaining comes from a 130-fold enhancement in the normalized gradient of the field intensity (viz., the gradient of the field intensity divided by the field intensity). Also, the proposed structure offers roughly a 100-fold enhancement in the depth of the trapping potential. It is noteworthy that “self-induced back-action trapping” (SIBA), which has recently been the focus of interest in the context of optical resonators, does not take place in the proposed resonator. In this paper, we also point out some misconceptions about SIBA together with some hitherto unappreciated subtleties of the dipole approximation.
DOI
We propose a dielectric structure which focuses laser light well beyond the diffraction limit and thus considerably enhances the exerted optical trapping force upon dielectric nanoparticles. Although the structure supports a Fabry–Perot resonance, it actually acts as a nanoantenna in that the role of the resonance is to funnel the laser light into the structure. In comparison with the lens illuminating the structure, the proposed structure offers roughly a 10,000-fold enhancement in the trapping force—part of this enhancement comes from an 80-fold enhancement in the field intensity, whereas the remaining comes from a 130-fold enhancement in the normalized gradient of the field intensity (viz., the gradient of the field intensity divided by the field intensity). Also, the proposed structure offers roughly a 100-fold enhancement in the depth of the trapping potential. It is noteworthy that “self-induced back-action trapping” (SIBA), which has recently been the focus of interest in the context of optical resonators, does not take place in the proposed resonator. In this paper, we also point out some misconceptions about SIBA together with some hitherto unappreciated subtleties of the dipole approximation.
DOI
Stacks off tracks: a role for the golgin AtCASP in plant endoplasmic reticulum-Golgi apparatus tethering
Anne Osterrieder, Imogen A Sparkes, Stan W Botchway, Andy Ward, Tijs Ketelaar, Norbert de Ruijter, Chris Hawes
The plant Golgi apparatus modifies and sorts incoming proteins from the endoplasmic reticulum (ER) and synthesizes cell wall matrix material. Plant cells possess numerous motile Golgi bodies, which are connected to the ER by yet to be identified tethering factors. Previous studies indicated a role for cis-Golgi plant golgins, which are long coiled-coil domain proteins anchored to Golgi membranes, in Golgi biogenesis. Here we show a tethering role for the golgin AtCASP at the ER-Golgi interface. Using live-cell imaging, Golgi body dynamics were compared in Arabidopsis thaliana leaf epidermal cells expressing fluorescently tagged AtCASP, a truncated AtCASP-ΔCC lacking the coiled-coil domains, and the Golgi marker STtmd. Golgi body speed and displacement were significantly reduced in AtCASP-ΔCC lines. Using a dual-colour optical trapping system and a TIRF-tweezer system, individual Golgi bodies were captured in planta. Golgi bodies in AtCASP-ΔCC lines were easier to trap and the ER-Golgi connection was more easily disrupted. Occasionally, the ER tubule followed a trapped Golgi body with a gap, indicating the presence of other tethering factors. Our work confirms that the intimate ER-Golgi association can be disrupted or weakened by expression of truncated AtCASP-ΔCC and suggests that this connection is most likely maintained by a golgin-mediated tethering complex.
DOI
The plant Golgi apparatus modifies and sorts incoming proteins from the endoplasmic reticulum (ER) and synthesizes cell wall matrix material. Plant cells possess numerous motile Golgi bodies, which are connected to the ER by yet to be identified tethering factors. Previous studies indicated a role for cis-Golgi plant golgins, which are long coiled-coil domain proteins anchored to Golgi membranes, in Golgi biogenesis. Here we show a tethering role for the golgin AtCASP at the ER-Golgi interface. Using live-cell imaging, Golgi body dynamics were compared in Arabidopsis thaliana leaf epidermal cells expressing fluorescently tagged AtCASP, a truncated AtCASP-ΔCC lacking the coiled-coil domains, and the Golgi marker STtmd. Golgi body speed and displacement were significantly reduced in AtCASP-ΔCC lines. Using a dual-colour optical trapping system and a TIRF-tweezer system, individual Golgi bodies were captured in planta. Golgi bodies in AtCASP-ΔCC lines were easier to trap and the ER-Golgi connection was more easily disrupted. Occasionally, the ER tubule followed a trapped Golgi body with a gap, indicating the presence of other tethering factors. Our work confirms that the intimate ER-Golgi association can be disrupted or weakened by expression of truncated AtCASP-ΔCC and suggests that this connection is most likely maintained by a golgin-mediated tethering complex.
DOI
Endoplasmic reticulum and Golgi apparatus: old friends, novel intimate relationships
Alessandro Vitale, Emanuela Pedrazzini
The mid-nineteen century invention of subcellular fractionation and the application of electron microscopy to cell biology allowed us to discover the functional connections between the endoplasmic reticulum (ER) and Golgi apparatus in protein synthesis and secretion. This progress – which formed part of those steps forward resulting in the Nobel Prize for Physiology or Medicine in 1974 to Albert Claude, Christian de Duve and George Palade – opened the way to the discovery of intracellular membrane trafficking, the diverse compartments of the endomembrane system, and the secretory and endocytic pathways. The biosynthetic branch of the secretory pathway starts from the ER and leads to the Golgi apparatus as the first intermediate station. At the end of last century, the discovery of vesicle budding and fusion together with associated protein machinery, the continued refinement of electron microscopy, and the development of confocal microscopy and fluorescent protein tags – combining recombinant DNA and live imaging – have opened an intense and still-ongoing debate about the mechanistic aspects of the functional connections between compartments (Spang, 2013; Robinson et al., 2015). This is particularly important at ER exit, where thousands of proteins destined for secretion or different endomembrane compartments start their life.
DOI
The mid-nineteen century invention of subcellular fractionation and the application of electron microscopy to cell biology allowed us to discover the functional connections between the endoplasmic reticulum (ER) and Golgi apparatus in protein synthesis and secretion. This progress – which formed part of those steps forward resulting in the Nobel Prize for Physiology or Medicine in 1974 to Albert Claude, Christian de Duve and George Palade – opened the way to the discovery of intracellular membrane trafficking, the diverse compartments of the endomembrane system, and the secretory and endocytic pathways. The biosynthetic branch of the secretory pathway starts from the ER and leads to the Golgi apparatus as the first intermediate station. At the end of last century, the discovery of vesicle budding and fusion together with associated protein machinery, the continued refinement of electron microscopy, and the development of confocal microscopy and fluorescent protein tags – combining recombinant DNA and live imaging – have opened an intense and still-ongoing debate about the mechanistic aspects of the functional connections between compartments (Spang, 2013; Robinson et al., 2015). This is particularly important at ER exit, where thousands of proteins destined for secretion or different endomembrane compartments start their life.
DOI
Intraflagellar transport velocity is governed by the number of active KIF17 and KIF3AB motors and their motility properties under load
Bojan Milic, Johan O. L. Andreasson, Daniel W. Hogan, and Steven M. Block
Homodimeric KIF17 and heterotrimeric KIF3AB are processive, kinesin-2 family motors that act jointly to carry out anterograde intraflagellar transport (IFT), ferrying cargo along microtubules (MTs) toward the tips of cilia. How IFT trains attain speeds that exceed the unloaded rate of the slower, KIF3AB motor remains unknown. By characterizing the motility properties of kinesin-2 motors as a function of load we find that the increase in KIF3AB velocity, elicited by forward loads from KIF17 motors, cannot alone account for the speed of IFT trains in vivo. Instead, higher IFT velocities arise from an increased likelihood that KIF3AB motors dissociate from the MT, resulting in transport by KIF17 motors alone, unencumbered by opposition from KIF3AB. The rate of transport is therefore set by an equilibrium between a faster state, where only KIF17 motors move the train, and a slower state, where at least one KIF3AB motor on the train remains active in transport. The more frequently the faster state is accessed, the higher the overall velocity of the IFT train. We conclude that IFT velocity is governed by (i) the absolute numbers of each motor type on a given train, (ii) how prone KIF3AB is to dissociation from MTs relative to KIF17, and (iii) how prone both motors are to dissociation relative to binding MTs.
DOI
Homodimeric KIF17 and heterotrimeric KIF3AB are processive, kinesin-2 family motors that act jointly to carry out anterograde intraflagellar transport (IFT), ferrying cargo along microtubules (MTs) toward the tips of cilia. How IFT trains attain speeds that exceed the unloaded rate of the slower, KIF3AB motor remains unknown. By characterizing the motility properties of kinesin-2 motors as a function of load we find that the increase in KIF3AB velocity, elicited by forward loads from KIF17 motors, cannot alone account for the speed of IFT trains in vivo. Instead, higher IFT velocities arise from an increased likelihood that KIF3AB motors dissociate from the MT, resulting in transport by KIF17 motors alone, unencumbered by opposition from KIF3AB. The rate of transport is therefore set by an equilibrium between a faster state, where only KIF17 motors move the train, and a slower state, where at least one KIF3AB motor on the train remains active in transport. The more frequently the faster state is accessed, the higher the overall velocity of the IFT train. We conclude that IFT velocity is governed by (i) the absolute numbers of each motor type on a given train, (ii) how prone KIF3AB is to dissociation from MTs relative to KIF17, and (iii) how prone both motors are to dissociation relative to binding MTs.
DOI
Assembly of a functional and responsive microstructure by heat bonding of DNA-grafted colloidal brick
Yuki Sakamoto & Shoichi Toyabe
A micromachine constructed to possess various chemical and mechanical functions is one of the ultimate targets of technology. Conventional lithographic processes can be used to form complicated structures. However, they are basically limited to rigid and static structures with poor surface properties. Here, we demonstrate a novel method for assembling responsive and functional microstructures from diverse particles modified with DNA strands. The DNA strands are designed to form hairpins at room temperature and denature when heated. Structures are assembled through the simultaneous manipulation and heating of particles with “hot” optical tweezers, which incorporates the particles one by one. The flexible connection formed by DNA strands allows the responsive deformation of the structures with local controllability of the structural flexibility. We assembled a microscopic robot arm actuated by an external magnet, a hinge structure with a locally controlled connection flexibility and a three-dimensional double helix structure. The method is simple and can also be applied to build complex biological tissues from cells.
DOI
A micromachine constructed to possess various chemical and mechanical functions is one of the ultimate targets of technology. Conventional lithographic processes can be used to form complicated structures. However, they are basically limited to rigid and static structures with poor surface properties. Here, we demonstrate a novel method for assembling responsive and functional microstructures from diverse particles modified with DNA strands. The DNA strands are designed to form hairpins at room temperature and denature when heated. Structures are assembled through the simultaneous manipulation and heating of particles with “hot” optical tweezers, which incorporates the particles one by one. The flexible connection formed by DNA strands allows the responsive deformation of the structures with local controllability of the structural flexibility. We assembled a microscopic robot arm actuated by an external magnet, a hinge structure with a locally controlled connection flexibility and a three-dimensional double helix structure. The method is simple and can also be applied to build complex biological tissues from cells.
DOI
Wednesday, September 20, 2017
Unrevealed part of myosin's powerstroke accounts for high efficiency of muscle contraction
András Bibó, György Károlyi, Mihály Kovács
Myosin II, the motor protein driving muscle contraction, uses energy of ATP hydrolysis to produce movement along actin. The key step of energy transduction is the powerstroke, involving rotation of myosin's lever while myosin is attached to actin. Macroscopic measurements indicated high thermodynamic efficiency for energy conversion. However, single-molecule experiments indicated lower efficiency, provoking a long-standing discrepancy. Based on the Fluctuation-Dissipation Theorem, we built a sufficiently detailed but low degree-of-freedom model reconstructing the entire mechanoenzymatic cycle. We show that a high axial stiffness of the lever during an initial, experimentally yet unrevealed part of the powerstroke results in a short-time, ratchet-like Kramers effect, and is responsible for the missing efficiency. The second part of the powerstroke is an Eyring-like relaxation that dominantly contributes to lever rotation, but produces only a minor part of the work. The model reveals the structural background of myosin's capability to function as a robust molecular engine and a very precise load sensor as well. Our model also suggests an explanation for the malfunction of myosins harboring mutations that lead to hypertrophic cardiomyopathies with most severe clinical prognosis.
DOI
Myosin II, the motor protein driving muscle contraction, uses energy of ATP hydrolysis to produce movement along actin. The key step of energy transduction is the powerstroke, involving rotation of myosin's lever while myosin is attached to actin. Macroscopic measurements indicated high thermodynamic efficiency for energy conversion. However, single-molecule experiments indicated lower efficiency, provoking a long-standing discrepancy. Based on the Fluctuation-Dissipation Theorem, we built a sufficiently detailed but low degree-of-freedom model reconstructing the entire mechanoenzymatic cycle. We show that a high axial stiffness of the lever during an initial, experimentally yet unrevealed part of the powerstroke results in a short-time, ratchet-like Kramers effect, and is responsible for the missing efficiency. The second part of the powerstroke is an Eyring-like relaxation that dominantly contributes to lever rotation, but produces only a minor part of the work. The model reveals the structural background of myosin's capability to function as a robust molecular engine and a very precise load sensor as well. Our model also suggests an explanation for the malfunction of myosins harboring mutations that lead to hypertrophic cardiomyopathies with most severe clinical prognosis.
DOI
Measurement of viscosity of liquids using optical tweezers
Anna Statsenko, Wataru Inami, Yoshimasa Kawata
We propose a method for measuring viscosities of unknown liquids by using optical tweezers combined with optical microscopy. We trapped 1-μm particles in water–glycerin mixtures and analyzed the dependence of the motion on viscosity. Based on our calibration with various water–glycerin mixtures, we propose a method for determination of viscosities of unknown liquids with high accuracy. We discuss how the method can be applied to measure the viscosity of liquids that are available only in small quantities. This non-invasive method of studying viscosities could be especially applicable in investigations of biological samples.
DOI
We propose a method for measuring viscosities of unknown liquids by using optical tweezers combined with optical microscopy. We trapped 1-μm particles in water–glycerin mixtures and analyzed the dependence of the motion on viscosity. Based on our calibration with various water–glycerin mixtures, we propose a method for determination of viscosities of unknown liquids with high accuracy. We discuss how the method can be applied to measure the viscosity of liquids that are available only in small quantities. This non-invasive method of studying viscosities could be especially applicable in investigations of biological samples.
DOI
Reverse orbiting and spinning of a Rayleigh dielectric spheroid in a J0 Bessel optical beam
F. G. Mitri
Based on the electric dipole approximation, numerical computations for the optical orbital and spin radiation torques induced by a zeroth-order Bessel beam on a lossless dielectric subwavelength spheroid with arbitrary orientation in space are performed. The transverse optical force components are determined first, and then used to compute the longitudinal orbital torque component. Moreover, the Cartesian components of the spin radiation torque vector are evaluated. Numerical calculations illustrate the analysis with particular emphasis on the beam parameters, polarization of the magnetic vector potential forming the beam, and aspect ratio of the spheroid. The results demonstrate that the lossless dielectric subwavelength spheroid becomes irresponsive to the transfer of angular momentum, where the longitudinal orbital and spin radiation torque components vanish along singularity lines. Moreover, depending on the beam parameters and the spheroid location in space, the longitudinal orbital and spin torque components reverse sign, indicating a direction of rotation around the central axis of the beam and center of mass of the spheroid, respectively, in either the counterclockwise or clockwise directions. The results are important in particle dynamics and trapping applications, optical tweezers and spanners, and other related fields.
DOI
Based on the electric dipole approximation, numerical computations for the optical orbital and spin radiation torques induced by a zeroth-order Bessel beam on a lossless dielectric subwavelength spheroid with arbitrary orientation in space are performed. The transverse optical force components are determined first, and then used to compute the longitudinal orbital torque component. Moreover, the Cartesian components of the spin radiation torque vector are evaluated. Numerical calculations illustrate the analysis with particular emphasis on the beam parameters, polarization of the magnetic vector potential forming the beam, and aspect ratio of the spheroid. The results demonstrate that the lossless dielectric subwavelength spheroid becomes irresponsive to the transfer of angular momentum, where the longitudinal orbital and spin radiation torque components vanish along singularity lines. Moreover, depending on the beam parameters and the spheroid location in space, the longitudinal orbital and spin torque components reverse sign, indicating a direction of rotation around the central axis of the beam and center of mass of the spheroid, respectively, in either the counterclockwise or clockwise directions. The results are important in particle dynamics and trapping applications, optical tweezers and spanners, and other related fields.
DOI
Load Adaptation of Lamellipodial Actin Networks
Jan Mueller, Gregory Szep, Maria Nemethova, Ingrid de Vries, Arnon D. Lieber, Christoph Winkler, Karsten Kruse, J. Victor Small, Christian Schmeiser, Kinneret Keren, Robert Hauschild, Michael Sixt
Actin filaments polymerizing against membranes power endocytosis, vesicular traffic, and cell motility. In vitro reconstitution studies suggest that the structure and the dynamics of actin networks respond to mechanical forces. We demonstrate that lamellipodial actin of migrating cells responds to mechanical load when membrane tension is modulated. In a steady state, migrating cell filaments assume the canonical dendritic geometry, defined by Arp2/3-generated 70° branch points. Increased tension triggers a dense network with a broadened range of angles, whereas decreased tension causes a shift to a sparse configuration dominated by filaments growing perpendicularly to the plasma membrane. We show that these responses emerge from the geometry of branched actin: when load per filament decreases, elongation speed increases and perpendicular filaments gradually outcompete others because they polymerize the shortest distance to the membrane, where they are protected from capping. This network-intrinsic geometrical adaptation mechanism tunes protrusive force in response to mechanical load.
DOI
Actin filaments polymerizing against membranes power endocytosis, vesicular traffic, and cell motility. In vitro reconstitution studies suggest that the structure and the dynamics of actin networks respond to mechanical forces. We demonstrate that lamellipodial actin of migrating cells responds to mechanical load when membrane tension is modulated. In a steady state, migrating cell filaments assume the canonical dendritic geometry, defined by Arp2/3-generated 70° branch points. Increased tension triggers a dense network with a broadened range of angles, whereas decreased tension causes a shift to a sparse configuration dominated by filaments growing perpendicularly to the plasma membrane. We show that these responses emerge from the geometry of branched actin: when load per filament decreases, elongation speed increases and perpendicular filaments gradually outcompete others because they polymerize the shortest distance to the membrane, where they are protected from capping. This network-intrinsic geometrical adaptation mechanism tunes protrusive force in response to mechanical load.
DOI
Electrolyte-induced Instability of Colloidal Dispersions in Nonpolar Solvents
Gregory N. Smith, Samuel D. Finlayson, Sarah E. Rogers, Paul Bartlett, and Julian Eastoe
Dispersions of poly(methyl methacrylate) (PMMA) latexes were prepared in a low dielectric, nonpolar solvent (dodecane) both with and without the oil-soluble electrolyte, tetradodecylammonium-tetrakis(3,5-bis(trifluoromethyl)phenyl)borate. For dispersions with a high concentration of background electrolyte, the latexes become colloidally unstable and sediment in a short period of time (<1 h). This is completely reversible upon dilution. Instability of the dispersions is due to an apparent attraction between the colloids, directly observed using optical tweezers by bringing optically trapped particles into close proximity. Simple explanations generally used by colloid scientists to explain loss of stability (charge screening or stabilizer collapse) are insufficient to explain this observation. This unexpected interaction seems, therefore, to be a consequence of the materials that can be dispersed in low dielectric media and is expected to have ramifications for studying colloids in such solvents.
DOI
Dispersions of poly(methyl methacrylate) (PMMA) latexes were prepared in a low dielectric, nonpolar solvent (dodecane) both with and without the oil-soluble electrolyte, tetradodecylammonium-tetrakis(3,5-bis(trifluoromethyl)phenyl)borate. For dispersions with a high concentration of background electrolyte, the latexes become colloidally unstable and sediment in a short period of time (<1 h). This is completely reversible upon dilution. Instability of the dispersions is due to an apparent attraction between the colloids, directly observed using optical tweezers by bringing optically trapped particles into close proximity. Simple explanations generally used by colloid scientists to explain loss of stability (charge screening or stabilizer collapse) are insufficient to explain this observation. This unexpected interaction seems, therefore, to be a consequence of the materials that can be dispersed in low dielectric media and is expected to have ramifications for studying colloids in such solvents.
DOI
Monday, September 18, 2017
Accuracy and Mechanistic Details of Optical Printing of Single Au and Ag Nanoparticles
Julián Gargiulo, Ianina L. Violi, Santiago Cerrota, Lukáš Chvátal, Emiliano Cortés, Eduardo M. Perassi, Fernando Diaz, Pavel Zemánek, and Fernando D. Stefani
Optical printing is a powerful all-optical method that allows the incorporation of colloidal nanoparticles (NPs) onto substrates with nanometric precision. Here, we present a systematic study of the accuracy of optical printing of Au and Ag NPs, using different laser powers and wavelengths. When using light of wavelength tuned to the localized surface plasmon resonance (LSPR) of the NPs, the accuracy improves as the laser power is reduced, whereas for wavelengths off the LSPR, the accuracy is independent of the laser power. Complementary studies of the printing times of the NPs reveal the roles of Brownian and deterministic motion. Calculated trajectories of the NPs, taking into account the interplay between optical forces, electrostatic forces, and Brownian motion, allowed us to rationalize the experimental results and gain a detailed insight into the mechanism of the printing process. A clear framework is laid out for future optimizations of optical printing and optical manipulation of NPs near substrates.
DOI
Optical printing is a powerful all-optical method that allows the incorporation of colloidal nanoparticles (NPs) onto substrates with nanometric precision. Here, we present a systematic study of the accuracy of optical printing of Au and Ag NPs, using different laser powers and wavelengths. When using light of wavelength tuned to the localized surface plasmon resonance (LSPR) of the NPs, the accuracy improves as the laser power is reduced, whereas for wavelengths off the LSPR, the accuracy is independent of the laser power. Complementary studies of the printing times of the NPs reveal the roles of Brownian and deterministic motion. Calculated trajectories of the NPs, taking into account the interplay between optical forces, electrostatic forces, and Brownian motion, allowed us to rationalize the experimental results and gain a detailed insight into the mechanism of the printing process. A clear framework is laid out for future optimizations of optical printing and optical manipulation of NPs near substrates.
DOI
Rigorous full-wave calculation of optical forces on dielectric and metallic microparticles immersed in a vector Airy beam
Wanli Lu, Huajin Chen, Shiyang Liu, and Zhifang Lin
Based on the generalized Lorenz-Mie theory and the Maxwell stress tensor approach we present the first rigorous full-wave solution of the optical forces acting on spherical microparticles immersed in a two-dimensional vector Airy beam beyond the paraxial approximation. The critical aspect lies in evaluating efficiently and accurately the partial wave expansion coefficients of the incident Airy beam, which are achieved by using the vector angular spectrum representation for a variety of polarizations. The optical field distributions are then simulated to show the self-accelerating and self-healing effects of the Airy beam. The dielectric and gold microparticles are shown to be trapped within the main lobe or the nearby side-lobes mostly by the transverse gradient optical force while driven forward along the parabolic trajectory of the Airy beam by the longitudinal scattering force. It is thus demonstrated theoretically that the vector Airy beam has the capability of precisely transporting both dielectric and metallic microparticles along the prespecified curved paths.
DOI
Based on the generalized Lorenz-Mie theory and the Maxwell stress tensor approach we present the first rigorous full-wave solution of the optical forces acting on spherical microparticles immersed in a two-dimensional vector Airy beam beyond the paraxial approximation. The critical aspect lies in evaluating efficiently and accurately the partial wave expansion coefficients of the incident Airy beam, which are achieved by using the vector angular spectrum representation for a variety of polarizations. The optical field distributions are then simulated to show the self-accelerating and self-healing effects of the Airy beam. The dielectric and gold microparticles are shown to be trapped within the main lobe or the nearby side-lobes mostly by the transverse gradient optical force while driven forward along the parabolic trajectory of the Airy beam by the longitudinal scattering force. It is thus demonstrated theoretically that the vector Airy beam has the capability of precisely transporting both dielectric and metallic microparticles along the prespecified curved paths.
DOI
Mechanical measurement of hydrogen bonded host–guest systems under non-equilibrium, near-physiological conditions
Teresa Naranjo, Fernando Cerrón, Belén Nieto-Ortega, Alfonso Latorre, Álvaro Somoza, Borja Ibarra and Emilio M. Pérez
Decades after the birth of supramolecular chemistry, there are many techniques to measure noncovalent interactions, such as hydrogen bonding, under equilibrium conditions. As ensembles of molecules rapidly lose coherence, we cannot extrapolate bulk data to single-molecule events under non-equilibrium conditions, more relevant to the dynamics of biological systems. We present a new method that exploits the high force resolution of optical tweezers to measure at the single molecule level the mechanical strength of a hydrogen bonded host–guest pair out of equilibrium and under near-physiological conditions. We utilize a DNA reporter to unambiguously isolate single binding events. The Hamilton receptor–cyanuric acid host–guest system is used as a test bed. The force required to dissociate the host–guest system is ∼17 pN and increases with the pulling rate as expected for a system under non-equilibrium conditions. Blocking one of the hydrogen bonding sites results in a significant decrease of the force-to-break by 1–2 pN, pointing out the ability of the method to resolve subtle changes in the mechanical strength of the binding due to the individual H-bonding components. We believe the method will prove to be a versatile tool to address important questions in supramolecular chemistry.
DOI
Decades after the birth of supramolecular chemistry, there are many techniques to measure noncovalent interactions, such as hydrogen bonding, under equilibrium conditions. As ensembles of molecules rapidly lose coherence, we cannot extrapolate bulk data to single-molecule events under non-equilibrium conditions, more relevant to the dynamics of biological systems. We present a new method that exploits the high force resolution of optical tweezers to measure at the single molecule level the mechanical strength of a hydrogen bonded host–guest pair out of equilibrium and under near-physiological conditions. We utilize a DNA reporter to unambiguously isolate single binding events. The Hamilton receptor–cyanuric acid host–guest system is used as a test bed. The force required to dissociate the host–guest system is ∼17 pN and increases with the pulling rate as expected for a system under non-equilibrium conditions. Blocking one of the hydrogen bonding sites results in a significant decrease of the force-to-break by 1–2 pN, pointing out the ability of the method to resolve subtle changes in the mechanical strength of the binding due to the individual H-bonding components. We believe the method will prove to be a versatile tool to address important questions in supramolecular chemistry.
DOI
Force spectroscopy unravels the role of ionic strength on DNA-cisplatin interaction: Modulating the binding parameters
L. Oliveira and M. S. Rocha
In the present work we have gone a step forward in the understanding of the DNA-cisplatin interaction, investigating the role of the ionic strength on the complexes formation. To achieve this task, we use optical tweezers to perform force spectroscopy on the DNA-cisplatin complexes, determining their mechanical parameters as a function of the drug concentration in the sample for three different buffers. From such measurements, we determine the binding parameters and study their behavior as a function of the ionic strength. The equilibrium binding constant decreases with the counterion concentration ([Na]) and can be used to estimate the effective net charge of cisplatin in solution. The cooperativity degree of the binding reaction, on the other hand, increases with the ionic strength, as a result of the different conformational changes induced by the drug on the double-helix when binding under different buffer conditions. Such results can be used to modulate the drug binding to DNA, by appropriately setting the ionic strength of the surrounding buffer. The conclusions drawn provide significant new insights on the complex cooperative interactions between the DNA molecule and the class of platinum-based compounds, much used in chemotherapies.
DOI
In the present work we have gone a step forward in the understanding of the DNA-cisplatin interaction, investigating the role of the ionic strength on the complexes formation. To achieve this task, we use optical tweezers to perform force spectroscopy on the DNA-cisplatin complexes, determining their mechanical parameters as a function of the drug concentration in the sample for three different buffers. From such measurements, we determine the binding parameters and study their behavior as a function of the ionic strength. The equilibrium binding constant decreases with the counterion concentration ([Na]) and can be used to estimate the effective net charge of cisplatin in solution. The cooperativity degree of the binding reaction, on the other hand, increases with the ionic strength, as a result of the different conformational changes induced by the drug on the double-helix when binding under different buffer conditions. Such results can be used to modulate the drug binding to DNA, by appropriately setting the ionic strength of the surrounding buffer. The conclusions drawn provide significant new insights on the complex cooperative interactions between the DNA molecule and the class of platinum-based compounds, much used in chemotherapies.
DOI
Membrane Tension: A Challenging But Universal Physical Parameter in Cell Biology
BrunoPontes, Pascale Monzo Nils C.Gauthier
The plasma membrane separates the interior of cells from the outside environment. The membrane tension, defined as the force per unit length acting on a cross-section of membrane, regulates many vital biological processes. In this review, we summarize the first historical findings and the latest advances, showing membrane tension as an important physical parameter in cell biology. We also discuss how this parameter must be better integrated and we propose experimental approaches for key unanswered questions.
DOI
The plasma membrane separates the interior of cells from the outside environment. The membrane tension, defined as the force per unit length acting on a cross-section of membrane, regulates many vital biological processes. In this review, we summarize the first historical findings and the latest advances, showing membrane tension as an important physical parameter in cell biology. We also discuss how this parameter must be better integrated and we propose experimental approaches for key unanswered questions.
DOI
Thursday, September 14, 2017
Force-activated DNA substrates for probing individual proteins interacting with single-stranded DNA
Stephen R. Okoniewski Lyle Uyetake Thomas T. Perkins
Single-molecule force spectroscopy provides insight into how proteins bind to and move along DNA. Such studies often embed a single-stranded (ss) DNA region within a longer double-stranded (ds) DNA molecule. Yet, producing these substrates remains laborious and inefficient, particularly when using the traditional three-way hybridization. Here, we developed a force-activated substrate that yields an internal 1000 nucleotide (nt) ssDNA region when pulled partially into the overstretching transition (∼65 pN) by engineering a 50%-GC segment to have no adjacent GC base pairs. Once the template was made, these substrates were efficiently prepared by polymerase chain reaction amplification followed by site-specific nicking. We also generated a more complex structure used in high-resolution helicase studies, a DNA hairpin adjacent to 33 nt of ssDNA. The temporally defined generation of individual hairpin substrates in the presence of RecQ helicase and saturating adenine triphosphate let us deduce that RecQ binds to ssDNA via a near diffusion-limited reaction. More broadly, these substrates enable the precise initiation of an important class of protein–DNA interactions.
DOI
Single-molecule force spectroscopy provides insight into how proteins bind to and move along DNA. Such studies often embed a single-stranded (ss) DNA region within a longer double-stranded (ds) DNA molecule. Yet, producing these substrates remains laborious and inefficient, particularly when using the traditional three-way hybridization. Here, we developed a force-activated substrate that yields an internal 1000 nucleotide (nt) ssDNA region when pulled partially into the overstretching transition (∼65 pN) by engineering a 50%-GC segment to have no adjacent GC base pairs. Once the template was made, these substrates were efficiently prepared by polymerase chain reaction amplification followed by site-specific nicking. We also generated a more complex structure used in high-resolution helicase studies, a DNA hairpin adjacent to 33 nt of ssDNA. The temporally defined generation of individual hairpin substrates in the presence of RecQ helicase and saturating adenine triphosphate let us deduce that RecQ binds to ssDNA via a near diffusion-limited reaction. More broadly, these substrates enable the precise initiation of an important class of protein–DNA interactions.
DOI
Z-ring Structure and Constriction Dynamics in E. coli
Pramod Kumar, Amarjeet Yadav, Itzhak Fishov and Mario Feingold
The Z-ring plays a central role in bacterial division. It consists of FtsZ filaments, but the way these reorganize in the ring-like structure during septation remains largely unknown. Here, we measure the effective constriction dynamics of the ring. Using an oscillating optical trap, we can switch individual rod-shaped E. coli cells between horizontal and vertical orientations. In the vertical orientation, the fluorescent Z-ring image appears as a symmetric circular structure that renders itself to quantitative analysis. In the horizontal orientation, we use phase-contrast imaging to determine the extent of the cell constriction and obtain the effective time of division. We find evidence that the Z-ring constricts at a faster rate than the cell envelope such that its radial width (inwards from the cytoplasmic membrane) grows during septation. In this respect, our results differ from those recently obtained using photoactivated localization microscopy (PALM) where the radial width of the Z-ring was found to be approximately constant as the ring constricts. A possible reason for the different behavior of the constricting Z-rings could be the significant difference in the corresponding cell growth rates.
DOI
The Z-ring plays a central role in bacterial division. It consists of FtsZ filaments, but the way these reorganize in the ring-like structure during septation remains largely unknown. Here, we measure the effective constriction dynamics of the ring. Using an oscillating optical trap, we can switch individual rod-shaped E. coli cells between horizontal and vertical orientations. In the vertical orientation, the fluorescent Z-ring image appears as a symmetric circular structure that renders itself to quantitative analysis. In the horizontal orientation, we use phase-contrast imaging to determine the extent of the cell constriction and obtain the effective time of division. We find evidence that the Z-ring constricts at a faster rate than the cell envelope such that its radial width (inwards from the cytoplasmic membrane) grows during septation. In this respect, our results differ from those recently obtained using photoactivated localization microscopy (PALM) where the radial width of the Z-ring was found to be approximately constant as the ring constricts. A possible reason for the different behavior of the constricting Z-rings could be the significant difference in the corresponding cell growth rates.
DOI
Accurate nanoscale flexibility measurement of DNA and DNA–protein complexes by atomic force microscopy in liquid
Divakaran Murugesapillai, Serge Bouaziz, L. James Maher, III, Nathan E. Israeloff, Craig E. Cameron and Mark C. Williams
The elasticity of double-stranded DNA (dsDNA), as described by its persistence length, is critical for many biological processes, including genomic regulation. A persistence length value can be obtained using atomic force microscopy (AFM) imaging. However, most AFM studies have been done by depositing the sample on a surface using adhesive ligands and fitting the contour to a two-dimensional (2D) wormlike chain (WLC) model. This often results in a persistence length measurement that is different from the value determined using bulk and single molecule methods. We describe a method for obtaining accurate three-dimensional (3D) persistence length measurements for DNA and DNA–protein complexes by using a previously developed liquid AFM imaging method and then applying the 3D WLC model. To demonstrate the method, we image in both air and liquid several different dsDNA constructs and DNA–protein complexes that both increase (HIV-1 Vpr) and decrease (yeast HMO1) dsDNA persistence length. Fitting the liquid AFM-imaging contour to the 3D WLC model results in a value in agreement with measurements obtained in optical tweezers experiments. Because AFM also allows characterization of local DNA properties, the ability to correctly measure global flexibility will strongly increase the impact of measurements that use AFM imaging.
DOI
The elasticity of double-stranded DNA (dsDNA), as described by its persistence length, is critical for many biological processes, including genomic regulation. A persistence length value can be obtained using atomic force microscopy (AFM) imaging. However, most AFM studies have been done by depositing the sample on a surface using adhesive ligands and fitting the contour to a two-dimensional (2D) wormlike chain (WLC) model. This often results in a persistence length measurement that is different from the value determined using bulk and single molecule methods. We describe a method for obtaining accurate three-dimensional (3D) persistence length measurements for DNA and DNA–protein complexes by using a previously developed liquid AFM imaging method and then applying the 3D WLC model. To demonstrate the method, we image in both air and liquid several different dsDNA constructs and DNA–protein complexes that both increase (HIV-1 Vpr) and decrease (yeast HMO1) dsDNA persistence length. Fitting the liquid AFM-imaging contour to the 3D WLC model results in a value in agreement with measurements obtained in optical tweezers experiments. Because AFM also allows characterization of local DNA properties, the ability to correctly measure global flexibility will strongly increase the impact of measurements that use AFM imaging.
DOI
Parametric feedback cooling of levitated optomechanics in a parabolic mirror trap
Jamie Vovrosh, Muddassar Rashid, David Hempston, James Bateman, Mauro Paternostro, and Hendrik Ulbricht
Levitated optomechanics, a new experimental physics platform, holds promise for fundamental science and quantum technological sensing applications. We demonstrate a simple and robust geometry for optical trapping in vacuum of a single nanoparticle based on a parabolic mirror and the optical gradient force. We demonstrate parametric feedback cooling of all three motional degrees of freedom from room temperature to a few millikelvin. A single laser at 1550 nm and a single photodiode are used for trapping, position detection, and cooling for all three dimensions. Particles with diameters from 26 to 160 nm are trapped without feedback to 10−5 mbar10−5 mbar, and with feedback-engaged, the pressure is reduced to 10−6 mbar10−6 mbar. Modifications to the harmonic motion in the presence of noise and feedback are studied, and an experimental mechanical quality factor in excess of 4×1074×107 is evaluated. This particle manipulation is key to building a nanoparticle matter-wave interferometer in order to test the quantum superposition principle in the macroscopic domain.
DOI
Levitated optomechanics, a new experimental physics platform, holds promise for fundamental science and quantum technological sensing applications. We demonstrate a simple and robust geometry for optical trapping in vacuum of a single nanoparticle based on a parabolic mirror and the optical gradient force. We demonstrate parametric feedback cooling of all three motional degrees of freedom from room temperature to a few millikelvin. A single laser at 1550 nm and a single photodiode are used for trapping, position detection, and cooling for all three dimensions. Particles with diameters from 26 to 160 nm are trapped without feedback to 10−5 mbar10−5 mbar, and with feedback-engaged, the pressure is reduced to 10−6 mbar10−6 mbar. Modifications to the harmonic motion in the presence of noise and feedback are studied, and an experimental mechanical quality factor in excess of 4×1074×107 is evaluated. This particle manipulation is key to building a nanoparticle matter-wave interferometer in order to test the quantum superposition principle in the macroscopic domain.
DOI
Localized plasmonic structured illumination microscopy with optically trapped microlens
Anna Bezryadina, Jinxing Li, Junxiang Zhao, Alefia Kothambawala, Joseph Louis Ponsetto, Eric Huang, Joseph Wang and Zhaowei Liu
Localized plasmonic structured illumination microscopy (LPSIM) is a recently developed super resolution technique that demonstrates immense potential via arrays of localized plasmonic antennas. Microlens microscopy represents another distinct approach for improving resolution by introducing spherical lens with large refractive index to boost the effective numerical aperture of the imaging system. In this LetterPaper, we bridge together the LPSIM and optically trapped spherical microlenses, for the first time, to demonstrate a new super resolution technique for surface imaging. By trapping and moving polystyrene and TiO2 microspheres with optical tweezers on top of a LPSIM substrate, the new imaging system has achieved higher NA and resolution improvement.
DOI
Localized plasmonic structured illumination microscopy (LPSIM) is a recently developed super resolution technique that demonstrates immense potential via arrays of localized plasmonic antennas. Microlens microscopy represents another distinct approach for improving resolution by introducing spherical lens with large refractive index to boost the effective numerical aperture of the imaging system. In this LetterPaper, we bridge together the LPSIM and optically trapped spherical microlenses, for the first time, to demonstrate a new super resolution technique for surface imaging. By trapping and moving polystyrene and TiO2 microspheres with optical tweezers on top of a LPSIM substrate, the new imaging system has achieved higher NA and resolution improvement.
DOI
Wednesday, September 13, 2017
Effect of red light on optically trapped spermatozoa
Kay W. Chow, Daryl Preece, and Michael W. Berns
Successful artificial insemination relies on the use of high quality spermatozoa. One measure of sperm quality is swimming force. Increased swimming force has been correlated with higher sperm swimming speeds and improved reproductive success. It is hypothesized that by increasing sperm swimming speed, one can increase swimming force. Previous studies have shown that red light irradiation causes an increase in sperm swimming speed. In the current study, 633nm red light irradiation is shown to increase mean squared displacement in trapped sperm. The methodology allows for comparison of relative swimming forces between irradiated and non-irradiated samples.
DOI
Successful artificial insemination relies on the use of high quality spermatozoa. One measure of sperm quality is swimming force. Increased swimming force has been correlated with higher sperm swimming speeds and improved reproductive success. It is hypothesized that by increasing sperm swimming speed, one can increase swimming force. Previous studies have shown that red light irradiation causes an increase in sperm swimming speed. In the current study, 633nm red light irradiation is shown to increase mean squared displacement in trapped sperm. The methodology allows for comparison of relative swimming forces between irradiated and non-irradiated samples.
DOI
Roadmap for optofluidics
Paolo Minzioni, Roberto Osellame, Cinzia Sada, S Zhao, F G Omenetto, Kristinn B Gylfason, Tommy Haraldsson, Yibo Zhang, Aydogan Ozcan, Adam Wax
Optofluidics, nominally the research area where optics and fluidics merge, is a relatively new research field and it is only in the last decade that there has been a large increase in the number of optofluidic applications, as well as in the number of research groups, devoted to the topic. Nowadays optofluidics applications include, without being limited to, lab-on-a-chip devices, fluid-based and controlled lenses, optical sensors for fluids and for suspended particles, biosensors, imaging tools, etc. The long list of potential optofluidics applications, which have been recently demonstrated, suggests that optofluidic technologies will become more and more common in everyday life in the future, causing a significant impact on many aspects of our society. A characteristic of this research field, deriving from both its interdisciplinary origin and applications, is that in order to develop suitable solutions a combination of a deep knowledge in different fields, ranging from materials science to photonics, from microfluidics to molecular biology and biophysics, is often required. As a direct consequence, also being able to understand the long-term evolution of optofluidics research is not easy. In this article, we report several expert contributions on different topics so as to provide guidance for young scientists. At the same time, we hope that this document will also prove useful for funding institutions and stakeholders to better understand the perspectives and opportunities offered by this research field.
DOI
Optofluidics, nominally the research area where optics and fluidics merge, is a relatively new research field and it is only in the last decade that there has been a large increase in the number of optofluidic applications, as well as in the number of research groups, devoted to the topic. Nowadays optofluidics applications include, without being limited to, lab-on-a-chip devices, fluid-based and controlled lenses, optical sensors for fluids and for suspended particles, biosensors, imaging tools, etc. The long list of potential optofluidics applications, which have been recently demonstrated, suggests that optofluidic technologies will become more and more common in everyday life in the future, causing a significant impact on many aspects of our society. A characteristic of this research field, deriving from both its interdisciplinary origin and applications, is that in order to develop suitable solutions a combination of a deep knowledge in different fields, ranging from materials science to photonics, from microfluidics to molecular biology and biophysics, is often required. As a direct consequence, also being able to understand the long-term evolution of optofluidics research is not easy. In this article, we report several expert contributions on different topics so as to provide guidance for young scientists. At the same time, we hope that this document will also prove useful for funding institutions and stakeholders to better understand the perspectives and opportunities offered by this research field.
DOI
Size- and speed-dependent mechanical behavior in living mammalian cytoplasm
Jiliang Hu, Somaye Jafari, Yulong Han, Alan J. Grodzinsky, Shengqiang Cai, and Ming Guo
Active transport in the cytoplasm plays critical roles in living cell physiology. However, the mechanical resistance that intracellular compartments experience, which is governed by the cytoplasmic material property, remains elusive, especially its dependence on size and speed. Here we use optical tweezers to drag a bead in the cytoplasm and directly probe the mechanical resistance with varying size a and speed V. We introduce a method, combining the direct measurement and a simple scaling analysis, to reveal different origins of the size- and speed-dependent resistance in living mammalian cytoplasm. We show that the cytoplasm exhibits size-independent viscoelasticity as long as the effective strain rate V/a is maintained in a relatively low range (0.1 s−1 < V/a < 2 s−1) and exhibits size-dependent poroelasticity at a high effective strain rate regime (5 s−1 < V/a < 80 s−1). Moreover, the cytoplasmic modulus is found to be positively correlated with only V/a in the viscoelastic regime but also increases with the bead size at a constant V/a in the poroelastic regime. Based on our measurements, we obtain a full-scale state diagram of the living mammalian cytoplasm, which shows that the cytoplasm changes from a viscous fluid to an elastic solid, as well as from compressible material to incompressible material, with increases in the values of two dimensionless parameters, respectively. This state diagram is useful to understand the underlying mechanical nature of the cytoplasm in a variety of cellular processes over a broad range of speed and size scales.
DOI
Active transport in the cytoplasm plays critical roles in living cell physiology. However, the mechanical resistance that intracellular compartments experience, which is governed by the cytoplasmic material property, remains elusive, especially its dependence on size and speed. Here we use optical tweezers to drag a bead in the cytoplasm and directly probe the mechanical resistance with varying size a and speed V. We introduce a method, combining the direct measurement and a simple scaling analysis, to reveal different origins of the size- and speed-dependent resistance in living mammalian cytoplasm. We show that the cytoplasm exhibits size-independent viscoelasticity as long as the effective strain rate V/a is maintained in a relatively low range (0.1 s−1 < V/a < 2 s−1) and exhibits size-dependent poroelasticity at a high effective strain rate regime (5 s−1 < V/a < 80 s−1). Moreover, the cytoplasmic modulus is found to be positively correlated with only V/a in the viscoelastic regime but also increases with the bead size at a constant V/a in the poroelastic regime. Based on our measurements, we obtain a full-scale state diagram of the living mammalian cytoplasm, which shows that the cytoplasm changes from a viscous fluid to an elastic solid, as well as from compressible material to incompressible material, with increases in the values of two dimensionless parameters, respectively. This state diagram is useful to understand the underlying mechanical nature of the cytoplasm in a variety of cellular processes over a broad range of speed and size scales.
DOI
Kinetically Determined Hygroscopicity and Efflorescence of Sucrose–Ammonium Sulfate Aerosol Droplets under Lower Relative Humidity
Lin-Na Wang, Chen Cai, and Yun-Hong Zhang
Organic aerosols will likely form in semisolid, glassy, and high viscous state in the atmosphere, which show nonequilibrium kinetic characteristics at low relative humidity (RH) conditions. In this study, we applied optical tweezers to investigate the water transport in a sucrose/(NH4)2SO4 droplet with high organic to inorganic mole ratio (OIR). The characteristic time ratio between the droplet radius and the RH was used to describe the water mass transfer difference dependent on RH. For OIR greater than 1:1 in sucrose/(NH4)2SO4 droplets, the characteristic time ratio at low RH (<∼30% RH) was two orders magnitude greater than that at high RH (>∼60%). We also coupled vacuum FTIR spectrometer and a high-speed photography to study the efflorescence process in sucrose/(NH4)2SO4 droplets with low OIR. The crystalline fraction of (NH4)2SO4 was used to understand efflorescence behavior when the RH was linearly decreasing with a velocity of 1.2% RH min–1. Because of suppression of (NH4)2SO4 nucleation by addition of sucrose, the efflorescence relative humidity (ERH) of (NH4)2SO4 decrease from the range of ∼48.2% to ∼36.1% for pure (NH4)2SO4 droplets to from ∼44.7% to ∼25.4%, from ∼43.2% to ∼21.2%, and from ∼41.7% to ∼21.1% for the mixed droplets with OIR of 1:4, 1:3, and 1:2, respectively. No crystallization was observed when the OIR is higher than 1:1. Suppression of (NH4)2SO4 crystal growth was also observed under high viscous sucrose/(NH4)2SO4 droplets at lower RH.
DOI
Organic aerosols will likely form in semisolid, glassy, and high viscous state in the atmosphere, which show nonequilibrium kinetic characteristics at low relative humidity (RH) conditions. In this study, we applied optical tweezers to investigate the water transport in a sucrose/(NH4)2SO4 droplet with high organic to inorganic mole ratio (OIR). The characteristic time ratio between the droplet radius and the RH was used to describe the water mass transfer difference dependent on RH. For OIR greater than 1:1 in sucrose/(NH4)2SO4 droplets, the characteristic time ratio at low RH (<∼30% RH) was two orders magnitude greater than that at high RH (>∼60%). We also coupled vacuum FTIR spectrometer and a high-speed photography to study the efflorescence process in sucrose/(NH4)2SO4 droplets with low OIR. The crystalline fraction of (NH4)2SO4 was used to understand efflorescence behavior when the RH was linearly decreasing with a velocity of 1.2% RH min–1. Because of suppression of (NH4)2SO4 nucleation by addition of sucrose, the efflorescence relative humidity (ERH) of (NH4)2SO4 decrease from the range of ∼48.2% to ∼36.1% for pure (NH4)2SO4 droplets to from ∼44.7% to ∼25.4%, from ∼43.2% to ∼21.2%, and from ∼41.7% to ∼21.1% for the mixed droplets with OIR of 1:4, 1:3, and 1:2, respectively. No crystallization was observed when the OIR is higher than 1:1. Suppression of (NH4)2SO4 crystal growth was also observed under high viscous sucrose/(NH4)2SO4 droplets at lower RH.
DOI
A Single-Molecule View of Genome Editing Proteins: Biophysical Mechanisms for TALEs and CRISPR/Cas9
Luke Cuculis and Charles M. Schroeder
Exciting new advances in genome engineering have unlocked the potential to radically alter the treatment of human disease. In this review, we discuss the application of single-molecule techniques to uncover the mechanisms behind two premier classes of genome editing proteins: transcription activator-like effector nucleases (TALENs) and the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated system (Cas). These technologies have facilitated a striking number of gene editing applications in a variety of organisms; however, we are only beginning to understand the molecular mechanisms governing the DNA editing properties of these systems. Here, we discuss the DNA search and recognition process for TALEs and Cas9 that have been revealed by recent single-molecule experiments.
DOI
Exciting new advances in genome engineering have unlocked the potential to radically alter the treatment of human disease. In this review, we discuss the application of single-molecule techniques to uncover the mechanisms behind two premier classes of genome editing proteins: transcription activator-like effector nucleases (TALENs) and the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated system (Cas). These technologies have facilitated a striking number of gene editing applications in a variety of organisms; however, we are only beginning to understand the molecular mechanisms governing the DNA editing properties of these systems. Here, we discuss the DNA search and recognition process for TALEs and Cas9 that have been revealed by recent single-molecule experiments.
DOI
Monday, September 11, 2017
Light Scattering By Optically-Trapped Vesicles Affords Unprecedented Temporal Resolution Of Lipid-Raft Dynamics
Liam Collard, David Perez-Guaita, Bayan H. A. Faraj, Bayden R. Wood, Russell Wallis, Peter W. Andrew & Andrew J. Hudson
A spectroscopic technique is presented that is able to identify rapid changes in the bending modulus and fluidity of vesicle lipid bilayers on the micrometer scale, and distinguish between the presence and absence of heterogeneities in lipid-packing order. Individual unilamellar vesicles have been isolated using laser tweezers and, by measuring the intensity modulation of elastic back-scattered light, changes in the biophysical properties of lipid bilayers were revealed. Our approach offers unprecedented temporal resolution and, uniquely, physical transformations of lipid bilayers can be monitored on a length scale of micrometers. As an example, the deformation of a membrane bilayer following the gel-to-fluid phase transition in a pure phospholipid vesicle was observed to take place across an interval of 54 ± 5 ms corresponding to an estimated full-width of only ~1 m°C. Dynamic heterogeneities in packing order were detected in mixed-lipid bilayers. Using a ternary mixture of lipids, the modulated-intensity profile of elastic back-scattered light from an optically-trapped vesicle revealed an abrupt change in the bending modulus of the bilayer which could be associated with the dissolution of ordered microdomains (i.e., lipid rafts). This occurred across an interval of 30 ± 5 ms (equivalent to ~1 m°C).
DOI
A spectroscopic technique is presented that is able to identify rapid changes in the bending modulus and fluidity of vesicle lipid bilayers on the micrometer scale, and distinguish between the presence and absence of heterogeneities in lipid-packing order. Individual unilamellar vesicles have been isolated using laser tweezers and, by measuring the intensity modulation of elastic back-scattered light, changes in the biophysical properties of lipid bilayers were revealed. Our approach offers unprecedented temporal resolution and, uniquely, physical transformations of lipid bilayers can be monitored on a length scale of micrometers. As an example, the deformation of a membrane bilayer following the gel-to-fluid phase transition in a pure phospholipid vesicle was observed to take place across an interval of 54 ± 5 ms corresponding to an estimated full-width of only ~1 m°C. Dynamic heterogeneities in packing order were detected in mixed-lipid bilayers. Using a ternary mixture of lipids, the modulated-intensity profile of elastic back-scattered light from an optically-trapped vesicle revealed an abrupt change in the bending modulus of the bilayer which could be associated with the dissolution of ordered microdomains (i.e., lipid rafts). This occurred across an interval of 30 ± 5 ms (equivalent to ~1 m°C).
DOI
Optical trapping forces of a focused azimuthally polarized Bessel-Gaussian beam on a double-layered sphere
F. P. Wu, B. Zhang, Z. L. Liu, Y. Tang, N. Zhang
We calculate the trapping forces exerted by a highly focused Bessel-Gaussian beam on a double-layered sphere by means of vector diffraction integral, T-matrix method and Maxwell stress tensor integral. The Bessel-Gaussian beam is azimuthally polarized. Numerical results predicate that the double-layered sphere with air core can be stably trapped in three-dimensions. The trapping forces and efficiencies are dependent on the refraction index and size of the inner core. The trapping efficiency can be optimized by choosing the refraction indices of the inner core and outer layer. Our computational method can be easily modified for other laser beams and particles with arbitrary geometries and multilayers.
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We calculate the trapping forces exerted by a highly focused Bessel-Gaussian beam on a double-layered sphere by means of vector diffraction integral, T-matrix method and Maxwell stress tensor integral. The Bessel-Gaussian beam is azimuthally polarized. Numerical results predicate that the double-layered sphere with air core can be stably trapped in three-dimensions. The trapping forces and efficiencies are dependent on the refraction index and size of the inner core. The trapping efficiency can be optimized by choosing the refraction indices of the inner core and outer layer. Our computational method can be easily modified for other laser beams and particles with arbitrary geometries and multilayers.
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Charge Effects on the Efflorescence in Single Levitated Droplets
Gunter Hermann, Yan Zhang, Bernhard Wassermann, Henry Fischer, Marcel Quennet, and Eckart Rühl
The influence of electrical excess charges on the crystallization from supersaturated aqueous sodium chloride solutions is reported. This is accomplished by efflorescence studies on single levitated microdroplets using optical and electrodynamic levitation. Specifically, a strong increase in efflorescence humidity is observed as a function of the droplet’s negative excess charge, ranging up to −2.1 pC, with a distinct threshold behavior, increasing the relative efflorescence humidity, at which spontaneous nucleation occurs, from 44% for the neutral microparticle to 60%. These findings are interpreted by using molecular dynamics simulations for determining plausible structural patterns located near the particle surface that could serve as suitable precursors for the formation of critical clusters overcoming the nucleation barrier. These results, facilitating heterogeneous nucleation in the case of negatively charged microparticles, are compared to recent work on charge-induced nucleation of neat supercooled water, where a distinctly different nucleation behavior as a function of droplet charge has been observed.
DOI
The influence of electrical excess charges on the crystallization from supersaturated aqueous sodium chloride solutions is reported. This is accomplished by efflorescence studies on single levitated microdroplets using optical and electrodynamic levitation. Specifically, a strong increase in efflorescence humidity is observed as a function of the droplet’s negative excess charge, ranging up to −2.1 pC, with a distinct threshold behavior, increasing the relative efflorescence humidity, at which spontaneous nucleation occurs, from 44% for the neutral microparticle to 60%. These findings are interpreted by using molecular dynamics simulations for determining plausible structural patterns located near the particle surface that could serve as suitable precursors for the formation of critical clusters overcoming the nucleation barrier. These results, facilitating heterogeneous nucleation in the case of negatively charged microparticles, are compared to recent work on charge-induced nucleation of neat supercooled water, where a distinctly different nucleation behavior as a function of droplet charge has been observed.
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Vinculin forms a directionally asymmetric catch bond with F-actin
Derek L. Huang, Nicolas A. Bax, Craig D. Buckley, William I. Weis, Alexander R. Dunn
Vinculin is an actin-binding protein thought to reinforce cell-cell and cell-matrix adhesions. However, how mechanical load affects the vinculin–F-actin bond is unclear. Using a single-molecule optical trap assay, we found that vinculin forms a force-dependent catch bond with F-actin through its tail domain, but with lifetimes that depend strongly on the direction of the applied force. Force toward the pointed (–) end of the actin filament resulted in a bond that was maximally stable at 8 piconewtons, with a mean lifetime (12 seconds) 10 times as long as the mean lifetime when force was applied toward the barbed (+) end. A computational model of lamellipodial actin dynamics suggests that the directionality of the vinculin–F-actin bond could establish long-range order in the actin cytoskeleton. The directional and force-stabilized binding of vinculin to F-actin may be a mechanism by which adhesion complexes maintain front-rear asymmetry in migrating cells.
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Dynamics of microtubules: highlights of recent computational and experimental investigations
Valeri Barsegov, Jennifer Ross and Ruxandra Dima
Microtubules are found in most eukaryotic cells, with homologs in eubacteria and archea, and they have functional roles in mitosis, cell motility, intracellular transport, and the maintenance of cell shape. Numerous efforts have been expended over the last two decades to characterize the interactions between microtubules and the wide variety of microtubule associated proteins that control their dynamic behavior in cells resulting in microtubules being assembled and disassembled where and when they are required by the cell. We present the main findings regarding microtubule polymerization and depolymerization and review recent work about the molecular motors that modulate microtubule dynamics by inducing either microtubule depolymerization or severing. We also discuss the main experimental and computational approaches used to quantify the thermodynamics and mechanics of microtubule filaments.
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Microtubules are found in most eukaryotic cells, with homologs in eubacteria and archea, and they have functional roles in mitosis, cell motility, intracellular transport, and the maintenance of cell shape. Numerous efforts have been expended over the last two decades to characterize the interactions between microtubules and the wide variety of microtubule associated proteins that control their dynamic behavior in cells resulting in microtubules being assembled and disassembled where and when they are required by the cell. We present the main findings regarding microtubule polymerization and depolymerization and review recent work about the molecular motors that modulate microtubule dynamics by inducing either microtubule depolymerization or severing. We also discuss the main experimental and computational approaches used to quantify the thermodynamics and mechanics of microtubule filaments.
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Total momentum transfer produced by the photons of a multi-pass laser beam as an evident avenue for optical and mass metrology
Suren Vasilyan, Thomas Fröhlich, and Eberhard Manske
The use of the radiation pressure of a laser field, as an effect of the momentum transfer of the absorbed and re-emitted photons, suggests rather a complementary than an alternative possibility for metrology to generate calibration forces or to calibrate the optical power directly traceable to the International System of Units (SI). This paper reports a method and experimentally measured evidence on options to extend the effective use of radiation pressure for generating optical forces in the sub-microNewton (μN) range. Among other features and results presented, we emphasize the variability in controlling the accuracy of these forces through the proper utilization of the power of a multi-pass laser beam (semi- or completely) locked within confined systems. The direct measurements of these forces, augmented due to the partial or total momentum transfer of the photons of a multi-pass laser beam extended from several hundreds of picoNewton (pN) up to sub-μN range for the same power of laser source, are done by a precision force measurement system and compared with basic theoretical computations. We also discuss the opportunities to probe the fundamental physical limits associated with this method and to the considerable extent other competing contributing effects that might be regarded as sources of errors in metrological tasks.
DOI
The use of the radiation pressure of a laser field, as an effect of the momentum transfer of the absorbed and re-emitted photons, suggests rather a complementary than an alternative possibility for metrology to generate calibration forces or to calibrate the optical power directly traceable to the International System of Units (SI). This paper reports a method and experimentally measured evidence on options to extend the effective use of radiation pressure for generating optical forces in the sub-microNewton (μN) range. Among other features and results presented, we emphasize the variability in controlling the accuracy of these forces through the proper utilization of the power of a multi-pass laser beam (semi- or completely) locked within confined systems. The direct measurements of these forces, augmented due to the partial or total momentum transfer of the photons of a multi-pass laser beam extended from several hundreds of picoNewton (pN) up to sub-μN range for the same power of laser source, are done by a precision force measurement system and compared with basic theoretical computations. We also discuss the opportunities to probe the fundamental physical limits associated with this method and to the considerable extent other competing contributing effects that might be regarded as sources of errors in metrological tasks.
DOI
Wednesday, September 6, 2017
Pausing kinetics dominates strand-displacement polymerization by reverse transcriptase
Omri Malik, Hadeel Khamis, Sergei Rudnizky, Ailie Marx, Ariel Kaplan
Reverse transcriptase (RT) catalyzes the conversion of the viral RNA into an integration-competent double-stranded DNA, with a variety of enzymatic activities that include the ability to displace a non-template strand concomitantly with polymerization. Here, using high-resolution optical tweezers to follow the activity of the murine leukemia Virus RT, we show that strand-displacement polymerization is frequently interrupted. Abundant pauses are modulated by the strength of the DNA duplex ∼8 bp ahead, indicating the existence of uncharacterized RT/DNA interactions, and correspond to backtracking of the enzyme, whose recovery is also modulated by the duplex strength. Dissociation and reinitiation events, which induce long periods of inactivity and are likely the rate-limiting step in the synthesis of the genome in vivo, are modulated by the template structure and the viral nucleocapsid protein. Our results emphasize the potential regulatory role of conserved structural motifs, and may provide useful information for the development of potent and specific inhibitors.
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Reverse transcriptase (RT) catalyzes the conversion of the viral RNA into an integration-competent double-stranded DNA, with a variety of enzymatic activities that include the ability to displace a non-template strand concomitantly with polymerization. Here, using high-resolution optical tweezers to follow the activity of the murine leukemia Virus RT, we show that strand-displacement polymerization is frequently interrupted. Abundant pauses are modulated by the strength of the DNA duplex ∼8 bp ahead, indicating the existence of uncharacterized RT/DNA interactions, and correspond to backtracking of the enzyme, whose recovery is also modulated by the duplex strength. Dissociation and reinitiation events, which induce long periods of inactivity and are likely the rate-limiting step in the synthesis of the genome in vivo, are modulated by the template structure and the viral nucleocapsid protein. Our results emphasize the potential regulatory role of conserved structural motifs, and may provide useful information for the development of potent and specific inhibitors.
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Piconewton-Scale Analysis of Ras-BRaf Signal Transduction with Single-Molecule Force Spectroscopy
Chae-Seok Lim, Cheng Wen, Yanghui Sheng, Guangfu Wang, Zhuan Zhou, Shiqiang Wang, Huaye Zhang, Anpei Ye, J. Julius Zhu
Intermolecular interactions dominate the behavior of signal transduction in various physiological and pathological cell processes, yet assessing these interactions remains a challenging task. Here, this study reports a single-molecule force spectroscopic method that enables functional delineation of two interaction sites (≈35 pN and ≈90 pN) between signaling effectors Ras and BRaf in the canonical mitogen-activated protein kinase (MAPK) pathway. This analysis reveals mutations on BRaf at Q257 and A246, two sites frequently linked to cardio-faciocutaneous syndrome, result in ≈10−30 pN alterations in Ras[BOND]BRaf intermolecular binding force. The magnitude of changes in Ras[BOND]BRaf binding force correlates with the size of alterations in protein affinity and in α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-sensitive glutamate receptor (-R)-mediated synaptic transmission in neurons expressing replacement BRaf mutants, and predicts the extent of learning impairments in animals expressing replacement BRaf mutants. These results establish single-molecule force spectroscopy as an effective platform for evaluating the piconewton-level interaction of signaling molecules and predicting the behavior outcome of signal transduction.
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Intermolecular interactions dominate the behavior of signal transduction in various physiological and pathological cell processes, yet assessing these interactions remains a challenging task. Here, this study reports a single-molecule force spectroscopic method that enables functional delineation of two interaction sites (≈35 pN and ≈90 pN) between signaling effectors Ras and BRaf in the canonical mitogen-activated protein kinase (MAPK) pathway. This analysis reveals mutations on BRaf at Q257 and A246, two sites frequently linked to cardio-faciocutaneous syndrome, result in ≈10−30 pN alterations in Ras[BOND]BRaf intermolecular binding force. The magnitude of changes in Ras[BOND]BRaf binding force correlates with the size of alterations in protein affinity and in α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-sensitive glutamate receptor (-R)-mediated synaptic transmission in neurons expressing replacement BRaf mutants, and predicts the extent of learning impairments in animals expressing replacement BRaf mutants. These results establish single-molecule force spectroscopy as an effective platform for evaluating the piconewton-level interaction of signaling molecules and predicting the behavior outcome of signal transduction.
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Higher-order micro-fiber modes for Escherichia coli manipulation using a tapered seven-core fiber
Qiangzhou Rong, Yi Zhou, Xunli Yin, Zhihua Shao, and Xueguang Qiao
Optical manipulation using optical micro- and nano-fibers has shown potential for controlling bacterial activities such as E. coli trapping, propelling, and binding. Most of these manipulations have been performed using the propagation of the fundamental mode through the fiber. However, along the maximum mode-intensity axis, the higher-order modes have longer evanescent field extensions and larger field amplitudes at the fiber waist than the fundamental mode, opening up new possibilities for manipulating E. coli bacteria. In this work, a compact seven-core fiber (SCF)-based micro-fiber/optical tweezers was demonstrated for trapping, propelling, and rotating E. coli bacteria using the excitation of higher-order modes. The diameter of the SCF taper was 4 µm at the taper waist, which was much larger than that of previous nano-fiber tweezers. The laser wavelength was tunable from 1500 nm to 1600 nm, simultaneously causing photophoretic force, gradient force, and scattering force. This work provides a new opportunity for better understanding optical manipulation using higher-order modes at the single-cell level.
DOI
Optical manipulation using optical micro- and nano-fibers has shown potential for controlling bacterial activities such as E. coli trapping, propelling, and binding. Most of these manipulations have been performed using the propagation of the fundamental mode through the fiber. However, along the maximum mode-intensity axis, the higher-order modes have longer evanescent field extensions and larger field amplitudes at the fiber waist than the fundamental mode, opening up new possibilities for manipulating E. coli bacteria. In this work, a compact seven-core fiber (SCF)-based micro-fiber/optical tweezers was demonstrated for trapping, propelling, and rotating E. coli bacteria using the excitation of higher-order modes. The diameter of the SCF taper was 4 µm at the taper waist, which was much larger than that of previous nano-fiber tweezers. The laser wavelength was tunable from 1500 nm to 1600 nm, simultaneously causing photophoretic force, gradient force, and scattering force. This work provides a new opportunity for better understanding optical manipulation using higher-order modes at the single-cell level.
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Understanding and Reducing Photothermal Forces for the Fabrication of Au Nanoparticle Dimers by Optical Printing
Julian Gargiulo, Thomas Brick, Ianina L. Violi, Facundo C. Herrera, Toshihiko Shibanuma, Pablo Albella, Félix G. Requejo, Emiliano Cortés, Stefan A. Maier, and Fernando D. Stefani
Optical printing holds great potential to enable the use of the vast variety of colloidal nanoparticles (NPs) in nano- and microdevices and circuits. By means of optical forces, it enables the direct assembly of NPs, one by one, onto specific positions of solid surfaces with great flexibility of pattern design and no need of previous surface patterning. However, for unclear causes it was not possible to print identical NPs closer to each other than 300 nm. Here, we show that the repulsion restricting the optical printing of close by NPs arises from light absorption by the printed NPs and subsequent local heating. By optimizing heat dissipation, it is possible to reduce the minimum separation between NPs. Using a reduced graphene oxide layer on a sapphire substrate, we demonstrate for the first time the optical printing of Au—Au NP dimers. Modeling the experiments considering optical, thermophoretic, and thermo-osmotic forces we obtain a detailed understanding and a clear pathway for the optical printing fabrication of complex nano structures and circuits based on connected colloidal NPs.
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Optical printing holds great potential to enable the use of the vast variety of colloidal nanoparticles (NPs) in nano- and microdevices and circuits. By means of optical forces, it enables the direct assembly of NPs, one by one, onto specific positions of solid surfaces with great flexibility of pattern design and no need of previous surface patterning. However, for unclear causes it was not possible to print identical NPs closer to each other than 300 nm. Here, we show that the repulsion restricting the optical printing of close by NPs arises from light absorption by the printed NPs and subsequent local heating. By optimizing heat dissipation, it is possible to reduce the minimum separation between NPs. Using a reduced graphene oxide layer on a sapphire substrate, we demonstrate for the first time the optical printing of Au—Au NP dimers. Modeling the experiments considering optical, thermophoretic, and thermo-osmotic forces we obtain a detailed understanding and a clear pathway for the optical printing fabrication of complex nano structures and circuits based on connected colloidal NPs.
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A DNA-centered explanation of the DNA polymerase translocation mechanism
J. Ricardo Arias-Gonzalez
DNA polymerase couples chemical energy to translocation along a DNA template with a specific directionality while it replicates genetic information. According to single-molecule manipulation experiments, the polymerase-DNA complex can work against loads greater than 50 pN. It is not known, on the one hand, how chemical energy is transduced into mechanical motion, accounting for such large forces on sub-nanometer steps, and, on the other hand, how energy consumption in fidelity maintenance integrates in this non-equilibrium cycle. Here, we propose a translocation mechanism that points to the flexibility of the DNA, including its overstretching transition, as the principal responsible for the DNA polymerase ratcheting motion. By using thermodynamic analyses, we then find that an external load hardly affects the fidelity of the copying process and, consequently, that translocation and fidelity maintenance are loosely coupled processes. The proposed translocation mechanism is compatible with single-molecule experiments, structural data and stereochemical details of the DNA-protein complex that is formed during replication, and may be extended to RNA transcription.
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DNA polymerase couples chemical energy to translocation along a DNA template with a specific directionality while it replicates genetic information. According to single-molecule manipulation experiments, the polymerase-DNA complex can work against loads greater than 50 pN. It is not known, on the one hand, how chemical energy is transduced into mechanical motion, accounting for such large forces on sub-nanometer steps, and, on the other hand, how energy consumption in fidelity maintenance integrates in this non-equilibrium cycle. Here, we propose a translocation mechanism that points to the flexibility of the DNA, including its overstretching transition, as the principal responsible for the DNA polymerase ratcheting motion. By using thermodynamic analyses, we then find that an external load hardly affects the fidelity of the copying process and, consequently, that translocation and fidelity maintenance are loosely coupled processes. The proposed translocation mechanism is compatible with single-molecule experiments, structural data and stereochemical details of the DNA-protein complex that is formed during replication, and may be extended to RNA transcription.
DOI
Tuesday, September 5, 2017
Control of nucleus positioning in mouse oocytes
Maria Almonacid, Marie-EmilieTerret, Marie-Hélène Verlhac
The position of the nucleus in a cell can instruct morphogenesis in some cases, conveying spatial and temporal information and abnormal nuclear positioning can lead to disease. In oocytes from worm, sea urchin, frog and some fish, nucleus position regulates embryo development, it marks the animal pole and in Drosophila it defines the future dorso-ventral axis of the embryo and of the adult body plan. However, in mammals, the oocyte nucleus is centrally located and does not instruct any future embryo axis. Yet an off-center nucleus correlates with poor outcome for mouse and human oocyte development. This is surprising since oocytes further undergo two extremely asymmetric divisions in terms of the size of the daughter cells (enabling polar body extrusion), requiring an off-centering of their chromosomes. In this review we address not only the bio-physical mechanism controlling nucleus positioning via an actin-mediated pressure gradient, but we also speculate on potential biological relevance of nuclear positioning in mammalian oocytes and early embryos.
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The position of the nucleus in a cell can instruct morphogenesis in some cases, conveying spatial and temporal information and abnormal nuclear positioning can lead to disease. In oocytes from worm, sea urchin, frog and some fish, nucleus position regulates embryo development, it marks the animal pole and in Drosophila it defines the future dorso-ventral axis of the embryo and of the adult body plan. However, in mammals, the oocyte nucleus is centrally located and does not instruct any future embryo axis. Yet an off-center nucleus correlates with poor outcome for mouse and human oocyte development. This is surprising since oocytes further undergo two extremely asymmetric divisions in terms of the size of the daughter cells (enabling polar body extrusion), requiring an off-centering of their chromosomes. In this review we address not only the bio-physical mechanism controlling nucleus positioning via an actin-mediated pressure gradient, but we also speculate on potential biological relevance of nuclear positioning in mammalian oocytes and early embryos.
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3D correlative single-cell imaging utilizing fluorescence and refractive index tomography
Mirjam Schürmann, Gheorghe Cojoc, Salvatore Girardo, Elke Ulbricht, Jochen Guck, Paul Müller
Cells alter the path of light, a fact that leads to well-known aberrations in single cell or tissue imaging. Optical diffraction tomography (ODT) measures the biophysical property that causes these aberrations, the refractive index (RI). ODT is complementary to fluorescence imaging and does not require any markers. The present study introduces RI and fluorescence tomography with optofluidic rotation (RAFTOR) of suspended cells, facilitating the segmentation of the 3D-correlated RI and fluorescence data for a quantitative interpretation of the nuclear RI. The technique is validated with cell phantoms and used to confirm a lower nuclear RI for HL60 cells. Furthermore, the nuclear inversion of adult mouse photoreceptor cells is observed in the RI distribution. The applications shown confirm predictions of previous studies and illustrate the potential of RAFTOR to improve our understanding of cells and tissues.
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Cells alter the path of light, a fact that leads to well-known aberrations in single cell or tissue imaging. Optical diffraction tomography (ODT) measures the biophysical property that causes these aberrations, the refractive index (RI). ODT is complementary to fluorescence imaging and does not require any markers. The present study introduces RI and fluorescence tomography with optofluidic rotation (RAFTOR) of suspended cells, facilitating the segmentation of the 3D-correlated RI and fluorescence data for a quantitative interpretation of the nuclear RI. The technique is validated with cell phantoms and used to confirm a lower nuclear RI for HL60 cells. Furthermore, the nuclear inversion of adult mouse photoreceptor cells is observed in the RI distribution. The applications shown confirm predictions of previous studies and illustrate the potential of RAFTOR to improve our understanding of cells and tissues.
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Moiré deflectometry-based position detection for optical tweezers
Ali Akbar Khorshad, S. Nader S. Reihani, and Mohammad Taghi Tavassoly
Optical tweezers have proven to be indispensable tools for pico-Newton range force spectroscopy. A quadrant photodiode (QPD) positioned at the back focal plane of an optical tweezers’ condenser is commonly used for locating the trapped object. In this Letter, for the first time, to the best of our knowledge, we introduce a moiré pattern-based detection method for optical tweezers. We show, both theoretically and experimentally, that this detection method could provide considerably better position sensitivity compared to the commonly used detection systems. For instance, position sensitivity for a trapped 2.17 μm polystyrene bead is shown to be 71% better than the commonly used QPD-based detection method. Our theoretical and experimental results are in good agreement.
DOI
Optical tweezers have proven to be indispensable tools for pico-Newton range force spectroscopy. A quadrant photodiode (QPD) positioned at the back focal plane of an optical tweezers’ condenser is commonly used for locating the trapped object. In this Letter, for the first time, to the best of our knowledge, we introduce a moiré pattern-based detection method for optical tweezers. We show, both theoretically and experimentally, that this detection method could provide considerably better position sensitivity compared to the commonly used detection systems. For instance, position sensitivity for a trapped 2.17 μm polystyrene bead is shown to be 71% better than the commonly used QPD-based detection method. Our theoretical and experimental results are in good agreement.
DOI
Thermal tuning of spectral emission from optically trapped liquid-crystal droplet resonators
Alexandr Jonáš, Zdeněk Pilát, Jan Ježek, Silvie Bernatová, Tomáš Fořt, Pavel Zemánek, Mehdi Aas, and Alper Kiraz
Surfactant-stabilized emulsion droplets of liquid crystals (LCs) suspended in water and labeled with a fluorescent dye form active, anisotropic optofluidic microresonators. These microresonators can host whispering gallery modes (WGMs), high-quality morphology-dependent optical resonances that are supported due to the contrast of refractive index between the LC droplets and the surrounding aqueous medium. In addition, owing to the refractive index contrast, such LC emulsion droplets can be stably trapped in three dimensions using optical tweezers, enabling long-term investigation of their spectral characteristics. We explore various combinations of fluorescently dyed LC droplets and host liquid-surfactant systems and show that the WGM emission spectra of optical resonators based on optically trapped LC emulsion droplets can be largely and (almost) reversibly tuned by controlled changes of the ambient temperature. Depending on the actual range of temperature modulation and LC phase of the studied droplet, thermally induced effects can either lead to phase transitions in the LC droplets or cause modifications of their refractive index profile without changing their LC phase. Our results indicate feasibility of this approach for creating miniature thermally tunable sources of coherent light that can be manipulated and stabilized by optical forces.
DOI
Surfactant-stabilized emulsion droplets of liquid crystals (LCs) suspended in water and labeled with a fluorescent dye form active, anisotropic optofluidic microresonators. These microresonators can host whispering gallery modes (WGMs), high-quality morphology-dependent optical resonances that are supported due to the contrast of refractive index between the LC droplets and the surrounding aqueous medium. In addition, owing to the refractive index contrast, such LC emulsion droplets can be stably trapped in three dimensions using optical tweezers, enabling long-term investigation of their spectral characteristics. We explore various combinations of fluorescently dyed LC droplets and host liquid-surfactant systems and show that the WGM emission spectra of optical resonators based on optically trapped LC emulsion droplets can be largely and (almost) reversibly tuned by controlled changes of the ambient temperature. Depending on the actual range of temperature modulation and LC phase of the studied droplet, thermally induced effects can either lead to phase transitions in the LC droplets or cause modifications of their refractive index profile without changing their LC phase. Our results indicate feasibility of this approach for creating miniature thermally tunable sources of coherent light that can be manipulated and stabilized by optical forces.
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Directional Optical Sorting of Silicon Nanoparticles
Daniil A. Shilkin, Evgeny V. Lyubin, Maxim R. Shcherbakov, Mikhail Lapine, and Andrey A. Fedyanin
Optical manipulation of nanoparticles is a topic of great practical importance, with applications in surface science, colloidal chemistry, microfluidics, biochemistry, and medicine. One of the major highlights of this topic is particle sorting, which serves to create monodisperse systems remotely and to separate particles of different composition and size. Here, we analyze optical forces acting on spherical silicon nanoparticles that exhibit high-quality Mie resonances and demonstrate the potential of optical sorting methods for these systems. In particular, we propose multidirectional static sorting of nanoparticles using noncollinear beams with different wavelengths, which results in all-optical separation into an angular spectrum of sizes. We also validate the proposed methods by considering the operation in the presence of Brownian motion and in the evanescent wave configuration.
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Optical manipulation of nanoparticles is a topic of great practical importance, with applications in surface science, colloidal chemistry, microfluidics, biochemistry, and medicine. One of the major highlights of this topic is particle sorting, which serves to create monodisperse systems remotely and to separate particles of different composition and size. Here, we analyze optical forces acting on spherical silicon nanoparticles that exhibit high-quality Mie resonances and demonstrate the potential of optical sorting methods for these systems. In particular, we propose multidirectional static sorting of nanoparticles using noncollinear beams with different wavelengths, which results in all-optical separation into an angular spectrum of sizes. We also validate the proposed methods by considering the operation in the presence of Brownian motion and in the evanescent wave configuration.
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Experiment study and FEM simulation on erythrocytes under linear stretching of optical micromanipulation
Ying Liu, Huadong Song, Panpan Zhu, Hao Lu, and Qi Tang
The elasticity of erythrocytes is an important criterion to evaluate the quality of blood. This paper presents a novel research on erythrocytes’ elasticity with the application of optical tweezers and the finite element method (FEM) during blood storage. In this work, the erythrocytes with different in vitro times were linearly stretched by trapping force using optical tweezers and the time dependent elasticity of erythrocytes was investigated. The experimental results indicate that the membrane shear moduli of erythrocytes increased with the increasing in vitro time, namely the elasticity was decreasing. Simultaneously, an erythrocyte shell model with two parameters (membrane thickness h and membrane shear modulus H) was built to simulate the linear stretching states of erythrocytes by the FEM, and the simulations conform to the results obtained in the experiment. The evolution process was found that the erythrocytes membrane thicknesses were decreasing. The analysis assumes that the partial proteins and lipid bilayer of erythrocyte membrane were decomposed during the in vitro preservation of blood, which results in thin thickness, weak bending resistance, and losing elasticity of erythrocyte membrane. This study implies that the FEM can be employed to investigate the inward mechanical property changes of erythrocyte in different environments, which also can be a guideline for studying the erythrocyte mechanical state suffered from different diseases.
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The elasticity of erythrocytes is an important criterion to evaluate the quality of blood. This paper presents a novel research on erythrocytes’ elasticity with the application of optical tweezers and the finite element method (FEM) during blood storage. In this work, the erythrocytes with different in vitro times were linearly stretched by trapping force using optical tweezers and the time dependent elasticity of erythrocytes was investigated. The experimental results indicate that the membrane shear moduli of erythrocytes increased with the increasing in vitro time, namely the elasticity was decreasing. Simultaneously, an erythrocyte shell model with two parameters (membrane thickness h and membrane shear modulus H) was built to simulate the linear stretching states of erythrocytes by the FEM, and the simulations conform to the results obtained in the experiment. The evolution process was found that the erythrocytes membrane thicknesses were decreasing. The analysis assumes that the partial proteins and lipid bilayer of erythrocyte membrane were decomposed during the in vitro preservation of blood, which results in thin thickness, weak bending resistance, and losing elasticity of erythrocyte membrane. This study implies that the FEM can be employed to investigate the inward mechanical property changes of erythrocyte in different environments, which also can be a guideline for studying the erythrocyte mechanical state suffered from different diseases.
DOI
Monday, September 4, 2017
Mode conversion enables optical pulling force in photonic crystal waveguides
Tongtong Zhu, Andrey Novitsky, Yongyin Cao, M. R. C. Mahdy, Lin Wang, Fangkui Sun, Zehui Jiang, and Weiqiang Ding
We propose a robust scheme to achieve optical pulling force using the guiding modes supported in a hollow core double-mode photonic crystal waveguide instead of the structured optical beams in free space investigated earlier. The waveguide under consideration supports both the 0th order mode with a larger forward momentum and the 1st order mode with a smaller forward momentum. When the 1st order mode is launched, the scattering by the object inside the waveguide results in the conversion from the 1st order mode to the 0th order mode, thus creating the optical pulling force according to the conservation of linear momentum. We present the quantitative agreement between the results derived from the mode conversion analysis and those from rigorous simulation using the finite-difference in the time-domain numerical method. Importantly, the optical pulling scheme presented here is robust and broadband with naturally occurred lateral equilibriums and has a long manipulation range. Flexibilities of the current configuration make it valuable for the optical force tailoring and optical manipulation operation, especially in microfluidic channel systems.
Investigation on the photophoretic lift force acting upon particles under light irradiation
Shuangling Dong, Yafei Liu
Considering the characteristics of photophoresis in the actual process, the viewpoint that particles in fluids will experience photophoretic lift force is proposed. The force is related to the particle motion with photophoresis but different from the traditional photophoretic force. Analysis shows that there are mainly two factors contributing to the lift force, one stems from the variation in the distribution of light intensity, the other originates from the rotational motion of particles in a direction which is perpendicular to the optical axis. The expression of the photophoretic lift force has been given in the study. The impacts of the force have been analyzed and validated by comparing with previous experimental results.
DOI
Considering the characteristics of photophoresis in the actual process, the viewpoint that particles in fluids will experience photophoretic lift force is proposed. The force is related to the particle motion with photophoresis but different from the traditional photophoretic force. Analysis shows that there are mainly two factors contributing to the lift force, one stems from the variation in the distribution of light intensity, the other originates from the rotational motion of particles in a direction which is perpendicular to the optical axis. The expression of the photophoretic lift force has been given in the study. The impacts of the force have been analyzed and validated by comparing with previous experimental results.
DOI
Single-molecule imaging and manipulation of biomolecular machines and systems
Ryota Iino, Tatsuya Iida, Akihiko Nakamura, Ei-ichiro Saita, Huijuan You, Yasushi Sako
Biological molecular machines support various activities and behaviors of cells, such as energy production, signal transduction, growth, differentiation, and migration.
We provide an overview of single-molecule imaging methods involving both small and large probes used to monitor the dynamic motions of molecular machines in vitro (purified proteins) and in living cells, and single-molecule manipulation methods used to measure the forces, mechanical properties and responses of biomolecules. We also introduce several examples of single-molecule analysis, focusing primarily on motor proteins and signal transduction systems.
Single-molecule analysis is a powerful approach to unveil the operational mechanisms both of individual molecular machines and of systems consisting of many molecular machines.
DOI
Biological molecular machines support various activities and behaviors of cells, such as energy production, signal transduction, growth, differentiation, and migration.
We provide an overview of single-molecule imaging methods involving both small and large probes used to monitor the dynamic motions of molecular machines in vitro (purified proteins) and in living cells, and single-molecule manipulation methods used to measure the forces, mechanical properties and responses of biomolecules. We also introduce several examples of single-molecule analysis, focusing primarily on motor proteins and signal transduction systems.
Single-molecule analysis is a powerful approach to unveil the operational mechanisms both of individual molecular machines and of systems consisting of many molecular machines.
DOI
Exploring the Denatured State Ensemble by Single-Molecule Chemo-Mechanical Unfolding: The Effect of Force, Temperature, and Urea
Emily J.Guinn, SusanMarqusee
While it is widely appreciated that the denatured state of a protein is a heterogeneous conformational ensemble, there is still debate over how this ensemble changes with environmental conditions. Here, we use single-molecule chemo-mechanical unfolding, which combines force and urea using the optical tweezers, together with traditional protein unfolding studies to explore how perturbants commonly used to unfold proteins (urea, force, and temperature) affect the denatured-state ensemble. We compare the urea m-values, which report on the change in solvent accessible surface area for unfolding, to probe the denatured state as a function of force, temperature, and urea. We find that while the urea- and force-induced denatured states expose similar amounts of surface area, the denatured state at high temperature and low urea concentration is more compact. To disentangle these two effects, we use destabilizing mutations that shift the Tm and Cm. We find that the compaction of the denatured state is related to changing temperature as the different variants of acyl-coenzyme A binding protein have similar m-values when they are at the same temperature but different urea concentration. These results have important implications for protein folding and stability under different environmental conditions.
DOI
While it is widely appreciated that the denatured state of a protein is a heterogeneous conformational ensemble, there is still debate over how this ensemble changes with environmental conditions. Here, we use single-molecule chemo-mechanical unfolding, which combines force and urea using the optical tweezers, together with traditional protein unfolding studies to explore how perturbants commonly used to unfold proteins (urea, force, and temperature) affect the denatured-state ensemble. We compare the urea m-values, which report on the change in solvent accessible surface area for unfolding, to probe the denatured state as a function of force, temperature, and urea. We find that while the urea- and force-induced denatured states expose similar amounts of surface area, the denatured state at high temperature and low urea concentration is more compact. To disentangle these two effects, we use destabilizing mutations that shift the Tm and Cm. We find that the compaction of the denatured state is related to changing temperature as the different variants of acyl-coenzyme A binding protein have similar m-values when they are at the same temperature but different urea concentration. These results have important implications for protein folding and stability under different environmental conditions.
DOI
Optical Bessel beam illumination of a subwavelength prolate gold (Au) spheroid coated by a layer of plasmonic material: radiation force, spin and orbital torques
FG Mitri
The optical radiation force, spin and orbital torques exerted on a subwavelength prolate gold spheroid coated by a layer of plasmonic material with negative permittivity and illuminated by either a zeroth-order (non-vortex) or a first-order vector Bessel (vortex) beam are computed in the framework of the electric dipole approximation method. Calculations for the Cartesian components of the optical radiation force on a subwavelength spheroid with arbitrary orientation in space are performed, with emphasis on the order (or topological charge), half-cone angle of the beam, and the plasmonic layer thickness on- and off-resonance. A repulsive (pushing) force is predicted for the layered subwavelength prolate spheroid, on- and off-resonance along the direction of wave propagation. Moreover, the Cartesian components of the spin radiation torque are computed where a negative longitudinal spin torque component can arise, suggesting a rotational twist of the spheroid around its center of mass in either the counter-clockwise or the clockwise (negative) direction of spinning. In addition, the longitudinal component of the orbital radiation torque exhibits sign reversal, indicating a revolution around the beam axis in either the counter-clockwise or the clockwise directions. The results show that the plasmonic resonance strongly alters the force, spin and orbital torque components, causing major amplitude enhancements, signs twists, and complex distributions in the transverse plane.
DOI
The optical radiation force, spin and orbital torques exerted on a subwavelength prolate gold spheroid coated by a layer of plasmonic material with negative permittivity and illuminated by either a zeroth-order (non-vortex) or a first-order vector Bessel (vortex) beam are computed in the framework of the electric dipole approximation method. Calculations for the Cartesian components of the optical radiation force on a subwavelength spheroid with arbitrary orientation in space are performed, with emphasis on the order (or topological charge), half-cone angle of the beam, and the plasmonic layer thickness on- and off-resonance. A repulsive (pushing) force is predicted for the layered subwavelength prolate spheroid, on- and off-resonance along the direction of wave propagation. Moreover, the Cartesian components of the spin radiation torque are computed where a negative longitudinal spin torque component can arise, suggesting a rotational twist of the spheroid around its center of mass in either the counter-clockwise or the clockwise (negative) direction of spinning. In addition, the longitudinal component of the orbital radiation torque exhibits sign reversal, indicating a revolution around the beam axis in either the counter-clockwise or the clockwise directions. The results show that the plasmonic resonance strongly alters the force, spin and orbital torque components, causing major amplitude enhancements, signs twists, and complex distributions in the transverse plane.
DOI
Theoretical estimation of nonlinear optical force on dielectric spherical particles of arbitrary size under femtosecond pulsed excitation
Anita Devi and Arijit K. De
Experimental evidence indicates that high-repetition-rate ultrafast pulsed excitation is more efficient in optical trapping of dielectric nanoparticles as compared with continuous-wave excitation at the same average power. The physics behind the different nature of force under these two excitation conditions remained deceptive until quite recently when it was theoretically explained, in the dipole limit, as a combined effect of (1) repetitive instantaneous momentum transfer and (2) optical Kerr nonlinearity. The role of optical Kerr effect was theoretically studied for larger dielectric spherical particles, in the ray optics limit, also. However, a theoretical underpinning is yet to be established as to whether the effect of optical nonlinearity is omnipresent across different particle sizes, which we investigate here. Using localized approximation of generalized Lorenz-Mie theory, we theoretically analyze the nature of force (and potential) and provide a detailed comparative discussion between this generalized scattering formulation with dipole scattering formulation for dielectric nanoparticles.
DOI
Experimental evidence indicates that high-repetition-rate ultrafast pulsed excitation is more efficient in optical trapping of dielectric nanoparticles as compared with continuous-wave excitation at the same average power. The physics behind the different nature of force under these two excitation conditions remained deceptive until quite recently when it was theoretically explained, in the dipole limit, as a combined effect of (1) repetitive instantaneous momentum transfer and (2) optical Kerr nonlinearity. The role of optical Kerr effect was theoretically studied for larger dielectric spherical particles, in the ray optics limit, also. However, a theoretical underpinning is yet to be established as to whether the effect of optical nonlinearity is omnipresent across different particle sizes, which we investigate here. Using localized approximation of generalized Lorenz-Mie theory, we theoretically analyze the nature of force (and potential) and provide a detailed comparative discussion between this generalized scattering formulation with dipole scattering formulation for dielectric nanoparticles.
DOI
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