In addition to thermal noise, which is essential to promote conformational transitions in biopolymers, the cellular environment is replete with a spectrum of athermal fluctuations that are produced from a plethora of active processes. To understand the effect of athermal noise on biological processes, we studied how a small oscillatory force affects the thermally induced folding and unfolding transition of an RNA hairpin, whose response to constant tension had been investigated extensively in both theory and experiments. Strikingly, our molecular simulations performed under overdamped condition show that even at a high (low) tension that renders the hairpin (un)folding improbable, a weak external oscillatory force at a certain frequency can synchronously enhance the transition dynamics of RNA hairpin and increase the mean transition rate. Furthermore, the RNA dynamics can still discriminate a signal with resonance frequency even when the signal is mixed among other signals with nonresonant frequencies. In fact, our computational demonstration of thermally induced resonance in RNA hairpin dynamics is a direct realization of the phenomena called stochastic resonance and resonant activation. Our study, amenable to experimental tests using optical tweezers, is of great significance to the folding of biopolymers in vivo that are subject to the broad spectrum of cellular noises.
Saturday, September 29, 2012
Weak temporal signals can synchronize and accelerate the transition dynamics of biopolymers under tension
Won Kyu Kim, Changbong Hyeon, and Wokyung Sung
In addition to thermal noise, which is essential to promote conformational transitions in biopolymers, the cellular environment is replete with a spectrum of athermal fluctuations that are produced from a plethora of active processes. To understand the effect of athermal noise on biological processes, we studied how a small oscillatory force affects the thermally induced folding and unfolding transition of an RNA hairpin, whose response to constant tension had been investigated extensively in both theory and experiments. Strikingly, our molecular simulations performed under overdamped condition show that even at a high (low) tension that renders the hairpin (un)folding improbable, a weak external oscillatory force at a certain frequency can synchronously enhance the transition dynamics of RNA hairpin and increase the mean transition rate. Furthermore, the RNA dynamics can still discriminate a signal with resonance frequency even when the signal is mixed among other signals with nonresonant frequencies. In fact, our computational demonstration of thermally induced resonance in RNA hairpin dynamics is a direct realization of the phenomena called stochastic resonance and resonant activation. Our study, amenable to experimental tests using optical tweezers, is of great significance to the folding of biopolymers in vivo that are subject to the broad spectrum of cellular noises.
In addition to thermal noise, which is essential to promote conformational transitions in biopolymers, the cellular environment is replete with a spectrum of athermal fluctuations that are produced from a plethora of active processes. To understand the effect of athermal noise on biological processes, we studied how a small oscillatory force affects the thermally induced folding and unfolding transition of an RNA hairpin, whose response to constant tension had been investigated extensively in both theory and experiments. Strikingly, our molecular simulations performed under overdamped condition show that even at a high (low) tension that renders the hairpin (un)folding improbable, a weak external oscillatory force at a certain frequency can synchronously enhance the transition dynamics of RNA hairpin and increase the mean transition rate. Furthermore, the RNA dynamics can still discriminate a signal with resonance frequency even when the signal is mixed among other signals with nonresonant frequencies. In fact, our computational demonstration of thermally induced resonance in RNA hairpin dynamics is a direct realization of the phenomena called stochastic resonance and resonant activation. Our study, amenable to experimental tests using optical tweezers, is of great significance to the folding of biopolymers in vivo that are subject to the broad spectrum of cellular noises.
Optical sorting of particles by dual-channel line optical tweezers
Baiheng Ma, Baoli Yao, Fei Peng, Shaohui Yan, Ming Lei and Romano Rupp
A novel configuration of dual-channel line optical tweezers with a 'Y' shape is constructed for sorting of particles within a microfluidic chip. When yeast cells with different size pass the intersection of the specially designed line optical tweezers, they are separated and transported to different channels due to a difference in the forces exerted by the line tweezers that depends on the size of the cells. The influences of some experimental conditions, such as laser power and flow velocity, on the sorting efficiency are discussed.
DOI
A novel configuration of dual-channel line optical tweezers with a 'Y' shape is constructed for sorting of particles within a microfluidic chip. When yeast cells with different size pass the intersection of the specially designed line optical tweezers, they are separated and transported to different channels due to a difference in the forces exerted by the line tweezers that depends on the size of the cells. The influences of some experimental conditions, such as laser power and flow velocity, on the sorting efficiency are discussed.
DOI
Less than 5 Netrin-1 molecules initiate attraction but 200 Sema3A molecules are necessary for repulsion
Giulietta Pinato, Dan Cojoc, Linh Thuy Lien, Alessio Ansuini, Jelena Ban, Elisa D’Este, and Vincent Torre
Guidance molecules, such as Sema3A or Netrin-1, induce growth cone (GC) repulsion or attraction. In order to determine the speed of action and efficiency of these guidance cues we developed an experimental procedure to deliver controlled amounts of these molecules. Lipid vesicles encapsulating 10–104 molecules of Sema3A or Netrin-1 were manipulated with high spatial and temporal resolution by optical tweezers and their photolysis triggered by laser pulses. Guidance molecules released from the vesicles diffused and reached the GC membrane in a few seconds. Following their arrival, GCs retracted or grew in 20–120 s. By determining the number of guidance molecules trapped inside vesicles and estimating the fraction of guidance molecules reaching the GC, we show that the arrival of less than 5 Netrin-1 molecules on the GC membrane is sufficient to induce growth. In contrast, the arrival of about 200 Sema3A molecules is necessary to induce filopodia repulsion.
DOI
Guidance molecules, such as Sema3A or Netrin-1, induce growth cone (GC) repulsion or attraction. In order to determine the speed of action and efficiency of these guidance cues we developed an experimental procedure to deliver controlled amounts of these molecules. Lipid vesicles encapsulating 10–104 molecules of Sema3A or Netrin-1 were manipulated with high spatial and temporal resolution by optical tweezers and their photolysis triggered by laser pulses. Guidance molecules released from the vesicles diffused and reached the GC membrane in a few seconds. Following their arrival, GCs retracted or grew in 20–120 s. By determining the number of guidance molecules trapped inside vesicles and estimating the fraction of guidance molecules reaching the GC, we show that the arrival of less than 5 Netrin-1 molecules on the GC membrane is sufficient to induce growth. In contrast, the arrival of about 200 Sema3A molecules is necessary to induce filopodia repulsion.
DOI
Wednesday, September 26, 2012
Dual-beam interference from a lensed multicore fiber and its application to optical trapping
Ashleigh L. Barron, Ajoy K. Kar, and Henry T. Bookey
A multicore all-fiber probe is demonstrated that has been fabricated using an electric arc fusion splicer. Interference of the fiber output when coherent light is coupled into two cores is investigated. The properties of the fringes created when the fiber is probing different media were found to be in general agreement with a beam propagation method simulation. Optical manipulation of microspheres near to the end of the probe is examined and the potential for controlled trapping explored. Polymer microspheres with diameters of 2 microns were formed into regular patterns due to the presence of the interference fringes.
DOI
A multicore all-fiber probe is demonstrated that has been fabricated using an electric arc fusion splicer. Interference of the fiber output when coherent light is coupled into two cores is investigated. The properties of the fringes created when the fiber is probing different media were found to be in general agreement with a beam propagation method simulation. Optical manipulation of microspheres near to the end of the probe is examined and the potential for controlled trapping explored. Polymer microspheres with diameters of 2 microns were formed into regular patterns due to the presence of the interference fringes.
DOI
Programmed −1 frameshifting efficiency correlates with RNA pseudoknot conformational plasticity, not resistance to mechanical unfolding
Dustin B. Ritchie, Daniel A. N. Foster, and Michael T. Woodside
Programmed −1 frameshifting, whereby the reading frame of a ribosome on messenger RNA is shifted in order to generate an alternate gene product, is often triggered by a pseudoknot structure in the mRNA in combination with an upstream slippery sequence. The efficiency of frameshifting varies widely for different sites, but the factors that determine frameshifting efficiency are not yet fully understood. Previous work has suggested that frameshifting efficiency is related to the resistance of the pseudoknot against mechanical unfolding. We tested this hypothesis by studying the mechanical properties of a panel of pseudoknots with frameshifting efficiencies ranging from 2% to 30%: four pseudoknots from retroviruses, two from luteoviruses, one from a coronavirus, and a nonframeshifting bacteriophage pseudoknot. Using optical tweezers to apply tension across the RNA, we measured the distribution of forces required to unfold each pseudoknot. We found that neither the average unfolding force, nor the unfolding kinetics, nor the parameters describing the energy landscape for mechanical unfolding of the pseudoknot (energy barrier height and distance to the transition state) could be correlated to frameshifting efficiency. These results indicate that the resistance of pseudoknots to mechanical unfolding is not a primary determinant of frameshifting efficiency. However, increased frameshifting efficiency was correlated with an increased tendency to form alternate, incompletely folded structures, suggesting a more complex picture of the role of the pseudoknot involving the conformational dynamics.
DOI
Programmed −1 frameshifting, whereby the reading frame of a ribosome on messenger RNA is shifted in order to generate an alternate gene product, is often triggered by a pseudoknot structure in the mRNA in combination with an upstream slippery sequence. The efficiency of frameshifting varies widely for different sites, but the factors that determine frameshifting efficiency are not yet fully understood. Previous work has suggested that frameshifting efficiency is related to the resistance of the pseudoknot against mechanical unfolding. We tested this hypothesis by studying the mechanical properties of a panel of pseudoknots with frameshifting efficiencies ranging from 2% to 30%: four pseudoknots from retroviruses, two from luteoviruses, one from a coronavirus, and a nonframeshifting bacteriophage pseudoknot. Using optical tweezers to apply tension across the RNA, we measured the distribution of forces required to unfold each pseudoknot. We found that neither the average unfolding force, nor the unfolding kinetics, nor the parameters describing the energy landscape for mechanical unfolding of the pseudoknot (energy barrier height and distance to the transition state) could be correlated to frameshifting efficiency. These results indicate that the resistance of pseudoknots to mechanical unfolding is not a primary determinant of frameshifting efficiency. However, increased frameshifting efficiency was correlated with an increased tendency to form alternate, incompletely folded structures, suggesting a more complex picture of the role of the pseudoknot involving the conformational dynamics.
DOI
Molecular Catch and Release: Controlled Delivery Using Optical Trapping with Light-Responsive Liposomes
Sarah J. Leung, Marek Romanowski
Gold-coated liposomes are maneuvered using an optical trap to achieve precise delivery of encapsulated molecular cargo. Movement and payload release from these plasmon resonant nanocapsules are independently controlled using a pulsed trapping beam. This technology enables in vitro delivery of a payload to a selected cell and may be applied to the interrogation of individual cells within their biological microenvironment.
DOI
Gold-coated liposomes are maneuvered using an optical trap to achieve precise delivery of encapsulated molecular cargo. Movement and payload release from these plasmon resonant nanocapsules are independently controlled using a pulsed trapping beam. This technology enables in vitro delivery of a payload to a selected cell and may be applied to the interrogation of individual cells within their biological microenvironment.
DOI
Tuesday, September 25, 2012
Cell Visco-Elasticity Measured with AFM and Optical Trapping at Sub-Micrometer Deformations
Schanila Nawaz, Paula Sánchez, Kai Bodensiek, Sai Li, Mikael Simons, Iwan A. T. Schaap
The measurement of the elastic properties of cells is widely used as an indicator for cellular changes during differentiation, upon drug treatment, or resulting from the interaction with the supporting matrix. Elasticity is routinely quantified by indenting the cell with a probe of an AFM while applying nano-Newton forces. Because the resulting deformations are in the micrometer range, the measurements will be affected by the finite thickness of the cell, viscous effects and even cell damage induced by the experiment itself. Here, we have analyzed the response of single 3T3 fibroblasts that were indented with a micrometer-sized bead attached to an AFM cantilever at forces from 30–600 pN, resulting in indentations ranging from 0.2 to 1.2 micrometer. To investigate the cellular response at lower forces up to 10 pN, we developed an optical trap to indent the cell in vertical direction, normal to the plane of the coverslip. Deformations of up to two hundred nanometers achieved at forces of up to 30 pN showed a reversible, thus truly elastic response that was independent on the rate of deformation. We found that at such small deformations, the elastic modulus of 100 Pa is largely determined by the presence of the actin cortex. At higher indentations, viscous effects led to an increase of the apparent elastic modulus. This viscous contribution that followed a weak power law, increased at larger cell indentations. Both AFM and optical trapping indentation experiments give consistent results for the cell elasticity. Optical trapping has the benefit of a lower force noise, which allows a more accurate determination of the absolute indentation. The combination of both techniques allows the investigation of single cells at small and large indentations and enables the separation of their viscous and elastic components.
DOI
The measurement of the elastic properties of cells is widely used as an indicator for cellular changes during differentiation, upon drug treatment, or resulting from the interaction with the supporting matrix. Elasticity is routinely quantified by indenting the cell with a probe of an AFM while applying nano-Newton forces. Because the resulting deformations are in the micrometer range, the measurements will be affected by the finite thickness of the cell, viscous effects and even cell damage induced by the experiment itself. Here, we have analyzed the response of single 3T3 fibroblasts that were indented with a micrometer-sized bead attached to an AFM cantilever at forces from 30–600 pN, resulting in indentations ranging from 0.2 to 1.2 micrometer. To investigate the cellular response at lower forces up to 10 pN, we developed an optical trap to indent the cell in vertical direction, normal to the plane of the coverslip. Deformations of up to two hundred nanometers achieved at forces of up to 30 pN showed a reversible, thus truly elastic response that was independent on the rate of deformation. We found that at such small deformations, the elastic modulus of 100 Pa is largely determined by the presence of the actin cortex. At higher indentations, viscous effects led to an increase of the apparent elastic modulus. This viscous contribution that followed a weak power law, increased at larger cell indentations. Both AFM and optical trapping indentation experiments give consistent results for the cell elasticity. Optical trapping has the benefit of a lower force noise, which allows a more accurate determination of the absolute indentation. The combination of both techniques allows the investigation of single cells at small and large indentations and enables the separation of their viscous and elastic components.
DOI
Physical manipulation of the Escherichia coli chromosome reveals its soft nature
James Pelletier, Ken Halvorsen, Bae-Yeun Ha, Raffaella Paparcone, Steven J. Sandler, Conrad L. Woldringh, Wesley P. Wong, and Suckjoon Jun
Replicating bacterial chromosomes continuously demix from each other and segregate within a compact volume inside the cell called the nucleoid. Although many proteins involved in this process have been identified, the nature of the global forces that shape and segregate the chromosomes has remained unclear because of limited knowledge of the micromechanical properties of the chromosome. In this work, we demonstrate experimentally the fundamentally soft nature of the bacterial chromosome and the entropic forces that can compact it in a crowded intracellular environment. We developed a unique “micropiston” and measured the force-compression behavior of single Escherichia colichromosomes in confinement. Our data show that forces on the order of 100 pN and free energies on the order of 105 kBT are sufficient to compress the chromosome to its in vivo size. For comparison, the pressure required to hold the chromosome at this size is a thousand-fold smaller than the surrounding turgor pressure inside the cell. Furthermore, by manipulation of molecular crowding conditions (entropic forces), we were able to observe in real time fast (approximately 10 s), abrupt, reversible, and repeatable compaction–decompaction cycles of individual chromosomes in confinement. In contrast, we observed much slower dissociation kinetics of a histone-like protein HU from the whole chromosome during its in vivo to in vitro transition. These results for the first time provide quantitative, experimental support for a physical model in which the bacterial chromosome behaves as a loaded entropic spring in vivo.
Replicating bacterial chromosomes continuously demix from each other and segregate within a compact volume inside the cell called the nucleoid. Although many proteins involved in this process have been identified, the nature of the global forces that shape and segregate the chromosomes has remained unclear because of limited knowledge of the micromechanical properties of the chromosome. In this work, we demonstrate experimentally the fundamentally soft nature of the bacterial chromosome and the entropic forces that can compact it in a crowded intracellular environment. We developed a unique “micropiston” and measured the force-compression behavior of single Escherichia colichromosomes in confinement. Our data show that forces on the order of 100 pN and free energies on the order of 105 kBT are sufficient to compress the chromosome to its in vivo size. For comparison, the pressure required to hold the chromosome at this size is a thousand-fold smaller than the surrounding turgor pressure inside the cell. Furthermore, by manipulation of molecular crowding conditions (entropic forces), we were able to observe in real time fast (approximately 10 s), abrupt, reversible, and repeatable compaction–decompaction cycles of individual chromosomes in confinement. In contrast, we observed much slower dissociation kinetics of a histone-like protein HU from the whole chromosome during its in vivo to in vitro transition. These results for the first time provide quantitative, experimental support for a physical model in which the bacterial chromosome behaves as a loaded entropic spring in vivo.
Cell polarisation and the immunological synapse
Karen L Angus, Gillian M Griffiths
Directed secretion by immune cells requires formation of the immunological synapse at the site of cell-cell contact, concomitant with a dramatic induction of cell polarity. Recent findings provide us with insights into the various steps that are required for these processes: for example, the first identification of a protein at the centrosome that regulates its relocation to the plasma membrane; the use of super-resolution imaging techniques to reveal a residual actin network at the immunological synapse that may permit secretory granule exocytosis; and the drawing of parallels between primary cilia and IS architecture. Here we discuss these and other novel findings that have advanced our understanding of the complex process of immunological synapse formation and subsequent induced cell polarity in immune cells.
Directed secretion by immune cells requires formation of the immunological synapse at the site of cell-cell contact, concomitant with a dramatic induction of cell polarity. Recent findings provide us with insights into the various steps that are required for these processes: for example, the first identification of a protein at the centrosome that regulates its relocation to the plasma membrane; the use of super-resolution imaging techniques to reveal a residual actin network at the immunological synapse that may permit secretory granule exocytosis; and the drawing of parallels between primary cilia and IS architecture. Here we discuss these and other novel findings that have advanced our understanding of the complex process of immunological synapse formation and subsequent induced cell polarity in immune cells.
Femtosecond-Pulsed Plasmonic Nanotweezers
Brian J. Roxworthy & Kimani C. Toussaint Jr.
We demonstrate for the first time plasmonic nanotweezers based on Au bowtie nanoantenna arrays (BNAs) that utilize a femtosecond-pulsed input source to enhance trapping of both Rayleigh and Mie particles. Using ultra-low input power densities, we demonstrate that the high-peak powers associated with a femtosecond source augment the trap stiffness to 2x that of nanotweezers employing a continuous-wave source, and 5x that of conventional tweezers using a femtosecond source. We show that for trapped fluorescent microparticles the two-photon response is enhanced by 2x in comparison to the response without nanoantennas. We also demonstrate tweezing of 80-nm diameter Ag nanoparticles, and observe an enhancement of the second-harmonic signal of ~3.5x for the combined nanoparticle-BNA system compared to the bare BNAs. Finally, under select illumination conditions, fusing of Ag nanoparticles to the BNAs is observed which holds potential forin situ fabrication of three-dimensional, bimetallic nanoantennas.
DOI
We demonstrate for the first time plasmonic nanotweezers based on Au bowtie nanoantenna arrays (BNAs) that utilize a femtosecond-pulsed input source to enhance trapping of both Rayleigh and Mie particles. Using ultra-low input power densities, we demonstrate that the high-peak powers associated with a femtosecond source augment the trap stiffness to 2x that of nanotweezers employing a continuous-wave source, and 5x that of conventional tweezers using a femtosecond source. We show that for trapped fluorescent microparticles the two-photon response is enhanced by 2x in comparison to the response without nanoantennas. We also demonstrate tweezing of 80-nm diameter Ag nanoparticles, and observe an enhancement of the second-harmonic signal of ~3.5x for the combined nanoparticle-BNA system compared to the bare BNAs. Finally, under select illumination conditions, fusing of Ag nanoparticles to the BNAs is observed which holds potential forin situ fabrication of three-dimensional, bimetallic nanoantennas.
DOI
Object-adapted optical trapping and shape-tracking of energy-switching helical bacteria
Matthias Koch & Alexander Rohrbach
Optical tweezers are a flexible manipulation tool used to grab micro-objects at a specific point, but a controlled manipulation of objects with more complex or changing shapes is hardly possible. Here, we demonstrate, by time-sharing optical forces, that it is possible to adapt the shape of the trapping potential to the shape of an elongated helical bacterium. In contrast to most other trapped objects, this structure can continuously change its helical shape (and therefore its mechanical energy), making trapping it much more difficult than trapping tiny non-living objects. The shape deformations of the only 200-nm-thin bacterium (Spiroplasma) are measured space-resolved at 800 Hz by exploiting local phase differences in coherently scattered trapping light. By localizing each slope of the bacterium we generate high-contrast, super-resolution movies in three dimensions, without any object staining. This approach will help in investigating the nanomechanics of single wall-less bacteria while reacting to external stimuli on a broad temporal bandwidth.
Monday, September 17, 2012
Combined versatile high-resolution optical tweezers and single-molecule fluorescence microscopy
George Sirinakis, Yuxuan Ren, Ying Gao, Zhiqun Xi, and Yongli Zhang
Optical trapping and single-molecule fluorescence are two major single-molecule approaches. Their combination has begun to show greater capability to study more complex systems than either method alone, but met many fundamental and technical challenges. We built an instrument that combines base-pair resolution dual-trap optical tweezers with single-molecule fluorescence microscopy. The instrument has complementary design and functionalities compared with similar microscopes previously described. The optical tweezers can be operated in constant force mode for easy data interpretation or in variable force mode for maximum spatiotemporal resolution. The single-molecule fluorescence detection can be implemented in either wide-field or confocal imaging configuration. To demonstrate the capabilities of the new instrument, we imaged a single stretched λ DNA molecule and investigated the dynamics of a DNA hairpin molecule in the presence of fluorophore-labeled complementary oligonucleotide. We simultaneously observed changes in the fluorescence signal and pauses in fast extension hopping of the hairpin due to association and dissociation of individual oligonucleotides. The combined versatile microscopy allows for greater flexibility to study molecular machines or assemblies at a single-molecule level.
DOI
Optical trapping and single-molecule fluorescence are two major single-molecule approaches. Their combination has begun to show greater capability to study more complex systems than either method alone, but met many fundamental and technical challenges. We built an instrument that combines base-pair resolution dual-trap optical tweezers with single-molecule fluorescence microscopy. The instrument has complementary design and functionalities compared with similar microscopes previously described. The optical tweezers can be operated in constant force mode for easy data interpretation or in variable force mode for maximum spatiotemporal resolution. The single-molecule fluorescence detection can be implemented in either wide-field or confocal imaging configuration. To demonstrate the capabilities of the new instrument, we imaged a single stretched λ DNA molecule and investigated the dynamics of a DNA hairpin molecule in the presence of fluorophore-labeled complementary oligonucleotide. We simultaneously observed changes in the fluorescence signal and pauses in fast extension hopping of the hairpin due to association and dissociation of individual oligonucleotides. The combined versatile microscopy allows for greater flexibility to study molecular machines or assemblies at a single-molecule level.
DOI
Thursday, September 13, 2012
Optical stirring in a droplet cell bioreactor
Murat Muradoglu, Thuong Le, Chun Yat Lau, Oi Wah Liew, and Tuck Wah Ng
In the context of a bioreactor, cells are sensitive to cues from other cells and mechanical stimuli from movement. The ability to provide the latter in a discrete fluidic system presents a significant challenge. From a prior finding that the location of the focus of a laser below particles relative to the beam axis producing a pushing effect in a predominant lateral sense, we advance an approach here that generates a gentle and tunable stirring effect. Computer simulation studies show that we are able to characterize this effect from the parameters that govern the optical forces and the movement of the particles. Experimental results with polystyrene microbeads and red blood cells confirm the notions from the simulations.
DOI
In the context of a bioreactor, cells are sensitive to cues from other cells and mechanical stimuli from movement. The ability to provide the latter in a discrete fluidic system presents a significant challenge. From a prior finding that the location of the focus of a laser below particles relative to the beam axis producing a pushing effect in a predominant lateral sense, we advance an approach here that generates a gentle and tunable stirring effect. Computer simulation studies show that we are able to characterize this effect from the parameters that govern the optical forces and the movement of the particles. Experimental results with polystyrene microbeads and red blood cells confirm the notions from the simulations.
DOI
Force sensing with a shaped dielectric micro-tool
D. B. Phillips, S. H. Simpson, J. A. Grieve, R. Bowman, G. M. Gibson, M. J. Padgett, J. G. Rarity, S. Hanna, M. J. Miles and D. M. Carberry
We analyse the thermal motion of a holographically trapped non-spherical force probe, capable of interrogating arbitrary samples with nanometer resolution. High speed video stereo-microscopy is used to track the translational and rotational coordinates of the micro-tool in three dimensions, and the complete 6 × 6 stiffness matrix for the system is determined using equipartition theorem. The Brownian motion of the extended structure is described in terms of a continuous distribution ofthermal ellipsoids. A centre of optical stress, at which rotational and translational motion is uncoupled, is observed and controlled. Once calibrated, the micro-tool is deployed in two modes of operation: as a force sensor with <150 femto-Newton sensitivity, and in a novel form of photonic force microscopy.
DOI
We analyse the thermal motion of a holographically trapped non-spherical force probe, capable of interrogating arbitrary samples with nanometer resolution. High speed video stereo-microscopy is used to track the translational and rotational coordinates of the micro-tool in three dimensions, and the complete 6 × 6 stiffness matrix for the system is determined using equipartition theorem. The Brownian motion of the extended structure is described in terms of a continuous distribution ofthermal ellipsoids. A centre of optical stress, at which rotational and translational motion is uncoupled, is observed and controlled. Once calibrated, the micro-tool is deployed in two modes of operation: as a force sensor with <150 femto-Newton sensitivity, and in a novel form of photonic force microscopy.
DOI
Wednesday, September 12, 2012
Optical forces in a non-diffracting vortex beam
Martin Šiler, Pavel Zemánek
Optical force acting upon a dielectric microparticle illuminated by a non-diffracting vortex beam is expressed using the Generalized Lorenz-Mie theory (GLMT). Numerical results are presented for different widths and topological charges of the vortex beam. We show that such particle may be stably trapped either in the dark center of the vortex beam, in one of two stable positions placed off the optical axis, and as the third option it may circulate along almost circular trajectory having its radius smaller or equal to the radius of the smallest high intensity vortex ring.
DOI
Optical force acting upon a dielectric microparticle illuminated by a non-diffracting vortex beam is expressed using the Generalized Lorenz-Mie theory (GLMT). Numerical results are presented for different widths and topological charges of the vortex beam. We show that such particle may be stably trapped either in the dark center of the vortex beam, in one of two stable positions placed off the optical axis, and as the third option it may circulate along almost circular trajectory having its radius smaller or equal to the radius of the smallest high intensity vortex ring.
DOI
Measuring intermolecular rupture forces with a combined TIRF-optical trap microscope and DNA curtains
Ja Yil Lee, Feng Wang, Teresa Fazio, Shalom Wind, Eric Greene
We report a new approach to probing DNA–protein interactions by combining optical tweezers with a high-throughput DNA curtains technique. Here we determine the forces required to remove the individual lipid-anchored DNA molecules from the bilayer. We demonstrate that DNA anchored to the bilayer through a single biotin–streptavidin linkage withstands ∼20 pN before being pulled free from the bilayer, whereas molecules anchored to the bilayer through multiple attachment points can withstand ⩾65 pN; access to this higher force regime is sufficient to probe the responses of protein–DNA interactions to force changes. As a proof-of-principle, we concurrently visualized DNA-bound fluorescently-tagged RNA polymerase while simultaneously stretching the DNA molecules. This work presents a step towards a powerful experimental platform that will enable concurrent visualization of DNA curtains while applying defined forces through optical tweezers.
DOI
We report a new approach to probing DNA–protein interactions by combining optical tweezers with a high-throughput DNA curtains technique. Here we determine the forces required to remove the individual lipid-anchored DNA molecules from the bilayer. We demonstrate that DNA anchored to the bilayer through a single biotin–streptavidin linkage withstands ∼20 pN before being pulled free from the bilayer, whereas molecules anchored to the bilayer through multiple attachment points can withstand ⩾65 pN; access to this higher force regime is sufficient to probe the responses of protein–DNA interactions to force changes. As a proof-of-principle, we concurrently visualized DNA-bound fluorescently-tagged RNA polymerase while simultaneously stretching the DNA molecules. This work presents a step towards a powerful experimental platform that will enable concurrent visualization of DNA curtains while applying defined forces through optical tweezers.
DOI
Creation of a controllable three dimensional optical chain by TEM01 mode radially polarized Laguerre–Gaussian beam
Jianwei Cao, Qingkui Chen, Hanming Guo
Three dimensional (3D) multi sites optical trapping requires multi focal spots in the focal region, which is not easy to achieve. In this paper, we present a novel design to create a controllable 3D optical chain that can stably trap and deliver particles purposely. On the basis of the vector diffraction theory, the complex pupil filters (CPF) is successfully designed to modulate the phase of TEM01 mode radially polarized Laguerre–Gaussian beams. With the optimized parameters of CPF, the 3D optical chains with two and five focal spots are created, respectively. Also, the 3D optical chain with five focal spots is periodical and moves forward (i.e., the direction far away the aplanatic system) with the increasing of the phase ψ of the outer zone of CPF. Moreover, the movement of the 3D optical chain can be well controlled by changing ψ purposely, which makes particle manipulation more controllable and flexible. This work is important for micromanipulation, micromachines, and microscopy.
Three dimensional (3D) multi sites optical trapping requires multi focal spots in the focal region, which is not easy to achieve. In this paper, we present a novel design to create a controllable 3D optical chain that can stably trap and deliver particles purposely. On the basis of the vector diffraction theory, the complex pupil filters (CPF) is successfully designed to modulate the phase of TEM01 mode radially polarized Laguerre–Gaussian beams. With the optimized parameters of CPF, the 3D optical chains with two and five focal spots are created, respectively. Also, the 3D optical chain with five focal spots is periodical and moves forward (i.e., the direction far away the aplanatic system) with the increasing of the phase ψ of the outer zone of CPF. Moreover, the movement of the 3D optical chain can be well controlled by changing ψ purposely, which makes particle manipulation more controllable and flexible. This work is important for micromanipulation, micromachines, and microscopy.
Focus shaping of linearly polarized Lorentz beam with sine-azimuthal variation wavefront
Xiumin Gao, Dawei Zhang, Mei Ting, Fu Rui, Qiufang Zhan, Songlin Zhuang
Focal shaping plays an important role in many optical focusing systems. In this paper, the focusing properties of linearly polarized Lorentz beam with sine-azimuthal variation wavefront were investigated. Simulation results show that the focal pattern can be altered considerably by the beam parameters and the phase parameter that indicates the phase change frequency on increasing azimuthal angle. And some novel focus shape may appear, including multiple-peak focal pattern, wheel focal pattern. And some optical gradient force distributions were also given to show that the focusing properties of this kind of Lorentz beams may be used to construct tunable optical traps, which may pave the way for application of laser diode in tweezers technique.
Focal shaping plays an important role in many optical focusing systems. In this paper, the focusing properties of linearly polarized Lorentz beam with sine-azimuthal variation wavefront were investigated. Simulation results show that the focal pattern can be altered considerably by the beam parameters and the phase parameter that indicates the phase change frequency on increasing azimuthal angle. And some novel focus shape may appear, including multiple-peak focal pattern, wheel focal pattern. And some optical gradient force distributions were also given to show that the focusing properties of this kind of Lorentz beams may be used to construct tunable optical traps, which may pave the way for application of laser diode in tweezers technique.
Tuesday, September 11, 2012
Single-molecule studies of RNAPII elongation
Jing Zhou, Volker Schweikhard, Steven M. Block
Elongation, the transcriptional phase in which RNA polymerase (RNAP) moves processively along a DNA template, occurs via a fundamental enzymatic mechanism that is thought to be universally conserved among multi-subunit polymerases in all kingdoms of life. Beyond this basic mechanism, a multitude of processes are integrated into transcript elongation, among them fidelity control, gene regulatory interactions involving elongation factors, RNA splicing or processing factors, and regulatory mechanisms associated with chromatin structure. Many kinetic and molecular details of the mechanism of the nucleotide addition cycle and its regulation, however, remain elusive and generate continued interest and even controversy. Recently, single-molecule approaches have emerged as powerful tools for the study of transcription in eukaryotic organisms. Here, we review recent progress and discuss some of the unresolved questions and ongoing debates, while anticipating future developments in the field. This article is part of a Special Issue entitled: RNA polymerase II Transcript Elongation.
DOI
Elongation, the transcriptional phase in which RNA polymerase (RNAP) moves processively along a DNA template, occurs via a fundamental enzymatic mechanism that is thought to be universally conserved among multi-subunit polymerases in all kingdoms of life. Beyond this basic mechanism, a multitude of processes are integrated into transcript elongation, among them fidelity control, gene regulatory interactions involving elongation factors, RNA splicing or processing factors, and regulatory mechanisms associated with chromatin structure. Many kinetic and molecular details of the mechanism of the nucleotide addition cycle and its regulation, however, remain elusive and generate continued interest and even controversy. Recently, single-molecule approaches have emerged as powerful tools for the study of transcription in eukaryotic organisms. Here, we review recent progress and discuss some of the unresolved questions and ongoing debates, while anticipating future developments in the field. This article is part of a Special Issue entitled: RNA polymerase II Transcript Elongation.
DOI
Microbead dynamics in optical trap assisted nanopatterning
Romain Fardel, Yu-Cheng Tsai and Craig B. Arnold
Optical near-field techniques allow one to overcome diffraction by positioning an optical element in close proximity to the surface of interest. In optical trap assisted nanopatterning, this optical element is a microbead optically trapped above the substrate in a liquid environment. Using high-speed microscopy, we show that under certain conditions, the laser pulse creates a gas bubble under the bead and that this bubble displaces the bead before disappearing. The bead then returns to its original position under the action of the scattering force of the optical trap. We measure the bead vertical trajectory and extract its terminal velocity in order to calculate the magnitude of the trapping force exerted on the bead. This work opens the way to a better understanding of the bead-surface interactions under laser irradiation and, therefore, contributes to the development of near-field techniques.
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Optical near-field techniques allow one to overcome diffraction by positioning an optical element in close proximity to the surface of interest. In optical trap assisted nanopatterning, this optical element is a microbead optically trapped above the substrate in a liquid environment. Using high-speed microscopy, we show that under certain conditions, the laser pulse creates a gas bubble under the bead and that this bubble displaces the bead before disappearing. The bead then returns to its original position under the action of the scattering force of the optical trap. We measure the bead vertical trajectory and extract its terminal velocity in order to calculate the magnitude of the trapping force exerted on the bead. This work opens the way to a better understanding of the bead-surface interactions under laser irradiation and, therefore, contributes to the development of near-field techniques.
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Direct observation of a force-induced switch in the anisotropic mechanical unfolding pathway of a protein
Bharat Jagannathan, Phillip J. Elms, Carlos Bustamante, and Susan Marqusee
Many biological processes generate force, and proteins have evolved to resist and respond to tension along different force axes. Single-molecule force spectroscopy allows for molecular insight into the behavior of proteins under force and the mechanism of protein folding in general. Here, we have used src SH3 to investigate the effect of different pulling axes under the low-force regime afforded by an optical trap. We find that this small cooperatively folded protein shows an anisotropic response to force; the protein is more mechanically resistant to force applied along a longitudinal axis compared to force applied perpendicular to the terminal β strand. In the longitudinal axis, we observe an unusual biphasic behavior revealing a force-induced switch in the unfolding mechanism suggesting the existence of two parallel unfolding pathways. A site-specific variant can selectively affect one of these pathways. Thus, even this simple two-state protein demonstrates a complex mechanical unfolding trajectory, accessing multiple unfolding pathways under the low-force regime of the optical trap; the specific unfolding pathway depends on the perturbation axis and the applied force.
Many biological processes generate force, and proteins have evolved to resist and respond to tension along different force axes. Single-molecule force spectroscopy allows for molecular insight into the behavior of proteins under force and the mechanism of protein folding in general. Here, we have used src SH3 to investigate the effect of different pulling axes under the low-force regime afforded by an optical trap. We find that this small cooperatively folded protein shows an anisotropic response to force; the protein is more mechanically resistant to force applied along a longitudinal axis compared to force applied perpendicular to the terminal β strand. In the longitudinal axis, we observe an unusual biphasic behavior revealing a force-induced switch in the unfolding mechanism suggesting the existence of two parallel unfolding pathways. A site-specific variant can selectively affect one of these pathways. Thus, even this simple two-state protein demonstrates a complex mechanical unfolding trajectory, accessing multiple unfolding pathways under the low-force regime of the optical trap; the specific unfolding pathway depends on the perturbation axis and the applied force.
Monday, September 10, 2012
Kinetics and Thermodynamics of Phenotype: Unwinding and Rewinding the Nucleosome
A.H. Mack, D.J. Schlingman, R.P. Ilagan, L. Regan, S.G.J. Mochrie
Chromatin “remodeling” is widely accepted as the mechanism that permits access to DNA by the transcription machinery. To date, however, there has been no experimental measurement of the changes in the kinetics and thermodynamics of the DNA–histone octamer association that are required to remodel chromatin so that transcription may occur. Here, we present the results of optical tweezer measurements that compare the kinetic and thermodynamic properties of nucleosomes composed of unmodified histones with those of nucleosomes that contain a mutant histone H4 (H4-R45H), which has been shown to allow SWI/SNF remodeling factor-independent transcription from the yeast HO promoter in vivo. Our measurements, carried out in a force-clamp mode, determine the force-dependent unwinding and rewinding rates of the nucleosome inner turn. At each force studied, nucleosomes containing H4-R45H unwind more rapidly and rewind more slowly than nucleosomes containing unmodified H4, indicating that the latter are the more stable. Extrapolation to forces at which the winding and unwinding rates are equal determines the absolute free energy of the nucleosome inner turn to be − 32kBT for nucleosomes containing unmodified H4 and − 27kBT for nucleosomes containing H4-R45H. Thus, the “loosening” or “remodeling” caused by this point mutation, which is demonstrated to be sufficient to allow transcriptional machinery access to the HOpromoter (in the absence of other remodeling factors), is 5kBT. The correlation between the free energy of the nucleosome inner turn and the sin (SWI/SNF-independent) transcription suggests that, beyond partial unwinding, complete histone unwinding may play a role in transcriptional activation.
DOI
Chromatin “remodeling” is widely accepted as the mechanism that permits access to DNA by the transcription machinery. To date, however, there has been no experimental measurement of the changes in the kinetics and thermodynamics of the DNA–histone octamer association that are required to remodel chromatin so that transcription may occur. Here, we present the results of optical tweezer measurements that compare the kinetic and thermodynamic properties of nucleosomes composed of unmodified histones with those of nucleosomes that contain a mutant histone H4 (H4-R45H), which has been shown to allow SWI/SNF remodeling factor-independent transcription from the yeast HO promoter in vivo. Our measurements, carried out in a force-clamp mode, determine the force-dependent unwinding and rewinding rates of the nucleosome inner turn. At each force studied, nucleosomes containing H4-R45H unwind more rapidly and rewind more slowly than nucleosomes containing unmodified H4, indicating that the latter are the more stable. Extrapolation to forces at which the winding and unwinding rates are equal determines the absolute free energy of the nucleosome inner turn to be − 32kBT for nucleosomes containing unmodified H4 and − 27kBT for nucleosomes containing H4-R45H. Thus, the “loosening” or “remodeling” caused by this point mutation, which is demonstrated to be sufficient to allow transcriptional machinery access to the HOpromoter (in the absence of other remodeling factors), is 5kBT. The correlation between the free energy of the nucleosome inner turn and the sin (SWI/SNF-independent) transcription suggests that, beyond partial unwinding, complete histone unwinding may play a role in transcriptional activation.
DOI
Compact Optical systems for Micromanipulation
Stefan Sinzinger, Andreas Oeder, Sebastian Stoebenau, Ronald Kampmann
Highly focused laser beams offer exciting possibilities for the handling and manipulation of small particles. Even though the technique of “optical tweezing” has been discussed and applied in research labs for more than 40 years a breakthrough in industrial applications has not taken place so far. Major obstacles result from the complex systems used to generate the necessary focus distribution. Our latest research indicates that optical tweezers based on compact and integrated optical systems are feasible. Such systems could open the field to a wide variety of new applications.
Highly focused laser beams offer exciting possibilities for the handling and manipulation of small particles. Even though the technique of “optical tweezing” has been discussed and applied in research labs for more than 40 years a breakthrough in industrial applications has not taken place so far. Major obstacles result from the complex systems used to generate the necessary focus distribution. Our latest research indicates that optical tweezers based on compact and integrated optical systems are feasible. Such systems could open the field to a wide variety of new applications.
Rotation of nanowires with radially higher-order Laguerre–Gaussian beams produced by computer-generated holograms
Li Shi, Jing Li, Tao Tao, and Xiaoping Wu
In this paper, the influence of radially higher index p of Laguerre–Gaussian (LG) beams on the rotation of nanowires is studied. Radially higher-order LG beams are produced by computer-generated holograms, which are displayed on a spatial light modulator. A series of experiments on manipulating ZnO nanowires was performed on our holographic optical tweezers platform. The experiments demonstrated that radially higher-order LG beams could effectively rotate nanowires along the innermost bright ring of themselves. Compared with radially low-order LG beams, they have larger torques exerted on nanowires and can make nanowires rotate more quickly.
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Optical stretching of giant unilamellar vesicles with an integrated dual-beam optical trap
Mehmet E. Solmaz, Roshni Biswas, Shalene Sankhagowit, James R. Thompson, Camilo A. Mejia, Noah Malmstadt, and Michelle L. Povinelli
We have integrated a dual-beam optical trap into a microfluidic platform and used it to study membrane mechanics in giant unilamellar vesicles (GUVs). We demonstrate the trapping and stretching of GUVs and characterize the membrane response to a step stress. We then measure area strain as a function of applied stress to extract the bending modulus of the lipid bilayer in the low-tension regime.
DOI
We have integrated a dual-beam optical trap into a microfluidic platform and used it to study membrane mechanics in giant unilamellar vesicles (GUVs). We demonstrate the trapping and stretching of GUVs and characterize the membrane response to a step stress. We then measure area strain as a function of applied stress to extract the bending modulus of the lipid bilayer in the low-tension regime.
DOI
Thursday, September 6, 2012
Application of Gaussian Optical Tweezers for Ultrafast Laser Assisted Direct–write Nanostructuring
Ulf QUENTIN, Karl-Heinz LEITZ, Ilya ALEXEEV, Michael SCHMIDT
The minimal size of optically generated structures is always affected by diffraction. This fundamental limitation does not allow laser radiation to be focussed much smaller than half of its wavelength and therefore it also limits minimal feature sizes of laser produced structures. In nearfield optics, however, the diffraction limit does not apply, which in principal allows for tighter
focussing. In this contribution, a flexible approach for direct-write nanostructuring is presented. Dielectric micro-particles are positioned by Gaussian optical tweezers and irradiated by ultrafast laser pulses. The particles focus the pulses and enable surface structuring.
The minimal size of optically generated structures is always affected by diffraction. This fundamental limitation does not allow laser radiation to be focussed much smaller than half of its wavelength and therefore it also limits minimal feature sizes of laser produced structures. In nearfield optics, however, the diffraction limit does not apply, which in principal allows for tighter
focussing. In this contribution, a flexible approach for direct-write nanostructuring is presented. Dielectric micro-particles are positioned by Gaussian optical tweezers and irradiated by ultrafast laser pulses. The particles focus the pulses and enable surface structuring.
Computational models for new fiber optic tweezers
R. S. Rodrigues Ribeiro, P. A. S. Jorge and A. Guerreiro
This paper discusses the calculation of the trapping forces in optical tweezers using a combination of the finite differences time domain (FDTD) method and the Lorentz force on electric dipoles. The results of 2D simulations of the trapping of a circular particle by a waveguide with a circular tip are presented and discussed.
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This paper discusses the calculation of the trapping forces in optical tweezers using a combination of the finite differences time domain (FDTD) method and the Lorentz force on electric dipoles. The results of 2D simulations of the trapping of a circular particle by a waveguide with a circular tip are presented and discussed.
DOI
An optical tweezer-based study of antimicrobial activity of silver nanoparticles
Yogesha, Sarbari Bhattacharya, M K Rabinal and Sharath Ananthamurthy
Understanding and characterizing microbial activity reduction in the presence of antimicrobial agents can help in the design and manufacture of antimicrobial drugs. We demonstrate the use of an optical tweezer setup in recording the changes in bacterial activity with time, induced by the presence of foreign bodies in a bacterial suspension. This is achieved by monitoring the fluctuations of an optically trapped polystyrene bead immersed in it. Examining the changes in the fluctuation pattern of the bead with time provides an accurate characterization of the reduction in the microbial activity. Here, we report on the effect of addition of silver nanoparticles on bacterial cultures of Pseudomonas aeroginosa, Escherichia coli and Bacillus subtilis. We observe a decrease in the bacterial activity with time for the investigated bacterial samples. This method in our opinion, enables one to track changes in bacterial activity levels as a function of time of contact with the antibacterial agent with greater efficacy than traditional cell counting methods.
Understanding and characterizing microbial activity reduction in the presence of antimicrobial agents can help in the design and manufacture of antimicrobial drugs. We demonstrate the use of an optical tweezer setup in recording the changes in bacterial activity with time, induced by the presence of foreign bodies in a bacterial suspension. This is achieved by monitoring the fluctuations of an optically trapped polystyrene bead immersed in it. Examining the changes in the fluctuation pattern of the bead with time provides an accurate characterization of the reduction in the microbial activity. Here, we report on the effect of addition of silver nanoparticles on bacterial cultures of Pseudomonas aeroginosa, Escherichia coli and Bacillus subtilis. We observe a decrease in the bacterial activity with time for the investigated bacterial samples. This method in our opinion, enables one to track changes in bacterial activity levels as a function of time of contact with the antibacterial agent with greater efficacy than traditional cell counting methods.
Tuesday, September 4, 2012
Electron spin resonance of nitrogen-vacancy centers in optically trapped nanodiamonds
Viva R. Horowitz, Benjamín J. Alemán, David J. Christle, Andrew N. Cleland, and David D. Awschalom
Using an optical tweezers apparatus, we demonstrate three-dimensional control of nanodiamonds in solution with simultaneous readout of ground-state electron-spin resonance (ESR) transitions in an ensemble of diamond nitrogen-vacancy color centers. Despite the motion and random orientation of nitrogen-vacancy centers suspended in the optical trap, we observe distinct peaks in the measured ESR spectra qualitatively similar to the same measurement in bulk. Accounting for the random dynamics, we model the ESR spectra observed in an externally applied magnetic field to enable dc magnetometry in solution. We estimate the dc magnetic field sensitivity based on variations in ESR line shapes to be approximately
. This technique may provide a pathway for spin-based magnetic, electric, and thermal sensing in fluidic environments and biophysical systems inaccessible to existing scanning probe techniques.
DOI
Using an optical tweezers apparatus, we demonstrate three-dimensional control of nanodiamonds in solution with simultaneous readout of ground-state electron-spin resonance (ESR) transitions in an ensemble of diamond nitrogen-vacancy color centers. Despite the motion and random orientation of nitrogen-vacancy centers suspended in the optical trap, we observe distinct peaks in the measured ESR spectra qualitatively similar to the same measurement in bulk. Accounting for the random dynamics, we model the ESR spectra observed in an externally applied magnetic field to enable dc magnetometry in solution. We estimate the dc magnetic field sensitivity based on variations in ESR line shapes to be approximately
. This technique may provide a pathway for spin-based magnetic, electric, and thermal sensing in fluidic environments and biophysical systems inaccessible to existing scanning probe techniques. DOI
Fluctuation-Mediated Optical Screening of Nanoparticles
Mamoru Tamura and Takuya Iida
Inspired by biological motors, we propose a guiding principle for selectively separating nanoparticles (NPs) by efficiently using the light-induced force (LIF) and thermal fluctuations. We demonstrate the possibility of transporting metallic NPs of different sizes with a size-selection accuracy of less than 10 nm even at room temperature by designing asymmetric spatiotemporal light fields. This technique will lead to unconventional nanoextraction processes based on light and fluctuations.
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Inspired by biological motors, we propose a guiding principle for selectively separating nanoparticles (NPs) by efficiently using the light-induced force (LIF) and thermal fluctuations. We demonstrate the possibility of transporting metallic NPs of different sizes with a size-selection accuracy of less than 10 nm even at room temperature by designing asymmetric spatiotemporal light fields. This technique will lead to unconventional nanoextraction processes based on light and fluctuations.
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An Annular Core Single Fiber Tweezers
Zhang, Yu; Liu, Zhihai; Yang, Jun; Yuan, Libo
An annular-core single fiber optical tweezers is proposed and fabricated by fiber grinding and polishing technology. With this optical tweezers probe, more than one yeast cell can be trapped simultaneously, besides that, a large polystyrene micro sphere with diameter about 45 μm can also be trapped. This new annular core single fiber tweezers can really achieve micro particle remote trapping and manipulation and more significantly, this new tweezers can trap multi particle or large particle by a single optical fiber probe.
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An annular-core single fiber optical tweezers is proposed and fabricated by fiber grinding and polishing technology. With this optical tweezers probe, more than one yeast cell can be trapped simultaneously, besides that, a large polystyrene micro sphere with diameter about 45 μm can also be trapped. This new annular core single fiber tweezers can really achieve micro particle remote trapping and manipulation and more significantly, this new tweezers can trap multi particle or large particle by a single optical fiber probe.
DOI
Ultrafast force-clamp spectroscopy of single molecules reveals load dependence of myosin working stroke
Marco Capitanio, Monica Canepari, Manuela Maffei, Diego Beneventi, Carina Monico, Francesco Vanzi, Roberto Bottinelli & Francesco Saverio Pavone
We describe a dual-trap force-clamp configuration that applies constant loads between a binding protein and an intermittently interacting biological polymer. The method has a measurement delay of only ~10 μs, allows detection of interactions as brief as ~100 μs and probes sub-nanometer conformational changes with a time resolution of tens of microseconds. We tested our method on molecular motors and DNA-binding proteins. We could apply constant loads to a single motor domain of myosin before its working stroke was initiated (0.2–1 ms), thus directly measuring its load dependence. We found that, depending on the applied load, myosin weakly interacted (<1 ms) with actin without production of movement, fully developed its working stroke or prematurely detached (<5 ms), thus reducing the working stroke size with load. Our technique extends single-molecule force-clamp spectroscopy and opens new avenues for investigating the effects of forces on biological processes.
DOI
We describe a dual-trap force-clamp configuration that applies constant loads between a binding protein and an intermittently interacting biological polymer. The method has a measurement delay of only ~10 μs, allows detection of interactions as brief as ~100 μs and probes sub-nanometer conformational changes with a time resolution of tens of microseconds. We tested our method on molecular motors and DNA-binding proteins. We could apply constant loads to a single motor domain of myosin before its working stroke was initiated (0.2–1 ms), thus directly measuring its load dependence. We found that, depending on the applied load, myosin weakly interacted (<1 ms) with actin without production of movement, fully developed its working stroke or prematurely detached (<5 ms), thus reducing the working stroke size with load. Our technique extends single-molecule force-clamp spectroscopy and opens new avenues for investigating the effects of forces on biological processes.
DOI
Monday, September 3, 2012
Rotational motions of optically trapped microscopic particles by vortex femtosecond laser
Ran Ling-Ling, Guo Zhong-Yi, Qu Shi-Liang
The rotational motions of the optically trapped microscopic particles by the vortex femtosecond laser beam are investigated in experiment. Black particles can be trapped and rotated by a vortex femtosecond laser beam very effectively because the vortex beam carries orbital angular momentum due to the helical wave-front structure associated with the central phase singularity. Trapped black particles rotate in the vortex beam due to the absorption of the angular momentum transferred from the vortex beam. The rotating directions of the trapped particles can be modulated by reversing the topological charge of the optical vortex in the vortex femtosecond beam. And the rotating speeds of the trapped microscopic particles depend on the topological charges of the vortex tweezer and the used pulse energies greatly.
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The rotational motions of the optically trapped microscopic particles by the vortex femtosecond laser beam are investigated in experiment. Black particles can be trapped and rotated by a vortex femtosecond laser beam very effectively because the vortex beam carries orbital angular momentum due to the helical wave-front structure associated with the central phase singularity. Trapped black particles rotate in the vortex beam due to the absorption of the angular momentum transferred from the vortex beam. The rotating directions of the trapped particles can be modulated by reversing the topological charge of the optical vortex in the vortex femtosecond beam. And the rotating speeds of the trapped microscopic particles depend on the topological charges of the vortex tweezer and the used pulse energies greatly.
DOI
Enhancing Single-Nanoparticle Surface-Chemistry by Plasmonic Overheating in an Optical Trap
Weihai Ni , Haojin Ba , Andrey A. Lutich , Frank Jäckel, and Jochen Feldmann
Surface-chemistry of individual, optically trapped plasmonic nanoparticles is modified and accelerated by plasmonic overheating. Depending on the optical trapping power, gold nanorods can exhibit red shifts of their plasmon resonance (i.e., increasing aspect ratio) under oxidative conditions. In contrast, in bulk exclusively blue shifts (decreasing aspect ratios) are observed. Supported by calculations, we explain this finding by local temperatures in the trap exceeding the boiling point of the solvent that cannot be achieved in bulk.
DOI
Surface-chemistry of individual, optically trapped plasmonic nanoparticles is modified and accelerated by plasmonic overheating. Depending on the optical trapping power, gold nanorods can exhibit red shifts of their plasmon resonance (i.e., increasing aspect ratio) under oxidative conditions. In contrast, in bulk exclusively blue shifts (decreasing aspect ratios) are observed. Supported by calculations, we explain this finding by local temperatures in the trap exceeding the boiling point of the solvent that cannot be achieved in bulk.
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Three-Dimensional Optical Trapping and Manipulation of Single Silver Nanowires
Zijie Yan , Justin Jureller , Julian Sweet , Mason Guffey , Matthew Pelton , and Norbert F. Scherer
We report the first experimental realization of all-optical trapping and manipulation of plasmonic nanowires in three dimensions. The optical beam used for trapping is the Fourier transform of a linearly polarized Bessel beam (termed FT-Bessel). The extended depth of focus of this beam enables the use of a retroreflection geometry to cancel radiation pressure in the beam propagation direction, making it possible to trap highly scattering and absorbing silver nanowires. Individual silver nanowires with lengths of several micrometers can be positioned by the trapping beam with precision better than 100 nm, and are oriented by the polarization of the trapping light with precision of approximately 1°. Multiple nanowires can be trapped simultaneously in spatially separated maxima of the trapping field. Since trapping in the interferometric FT-Bessel potential is robust in bulk solution and near surfaces, it will enable the controlled assembly of metal nanowires into plasmonic nanostructures.
DOI
We report the first experimental realization of all-optical trapping and manipulation of plasmonic nanowires in three dimensions. The optical beam used for trapping is the Fourier transform of a linearly polarized Bessel beam (termed FT-Bessel). The extended depth of focus of this beam enables the use of a retroreflection geometry to cancel radiation pressure in the beam propagation direction, making it possible to trap highly scattering and absorbing silver nanowires. Individual silver nanowires with lengths of several micrometers can be positioned by the trapping beam with precision better than 100 nm, and are oriented by the polarization of the trapping light with precision of approximately 1°. Multiple nanowires can be trapped simultaneously in spatially separated maxima of the trapping field. Since trapping in the interferometric FT-Bessel potential is robust in bulk solution and near surfaces, it will enable the controlled assembly of metal nanowires into plasmonic nanostructures.
DOI
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