Basudev Roy, Sudipta K. Bera, and Ayan Banerjee
We describe a simple yet powerful technique of simultaneously measuring both translational and rotational motion of mesoscopic particles in optical tweezers by measuring the backscattered intensity on a quadrant photodiode (QPD). While the measurement of translational motion by taking the difference of the backscattered intensity incident on adjacent quadrants of a QPD is well known, we demonstrate that rotational motion can be measured very precisely by taking the difference between the diagonal quadrants. The latter measurement eliminates the translational component entirely and leads to a detection sensitivity of around 50 mdeg at S/N of 2 for angular motion of a driven microrod. The technique is also able to resolve the translational and rotational Brownian motion components of the microrod in an unperturbed trap and can be very useful in measuring translation–rotation coupling of micro-objects induced by hydrodynamic interactions.
DOI
Friday, May 30, 2014
Contact Angle at the Leading Edge Controls Cell Protrusion Rate
Chiara Gabella, Elena Bertseva, Céline Bottier, Niccolò Piacentini, Alicia Bornert, Sylvia Jeney, László Forró, Ivo F. Sbalzarini, Jean-Jacques Meister, Alexander B. Verkhovsky
Plasma membrane tension and the pressure generated by actin polymerization are two antagonistic forces believed to define the protrusion rate at the leading edge of migrating cells [1, 2, 3, 4 and 5]. Quantitatively, resistance to actin protrusion is a product of membrane tension and mean local curvature (Laplace’s law); thus, it depends on the local geometry of the membrane interface. However, the role of the geometry of the leading edge in protrusion control has not been yet investigated. Here, we manipulate both the cell shape and substrate topography in the model system of persistently migrating fish epidermal keratocytes. We find that the protrusion rate does not correlate with membrane tension, but, instead, strongly correlates with cell roundness, and that the leading edge of the cell exhibits pinning on substrate ridges—a phenomenon characteristic of spreading of liquid drops. These results indicate that the leading edge could be considered a triple interface between the substrate, membrane, and extracellular medium and that the contact angle between the membrane and the substrate determines the load on actin polymerization and, therefore, the protrusion rate. Our findings thus illuminate a novel relationship between the 3D shape of the cell and its dynamics, which may have implications for cell migration in 3D environments.
DOI
Plasma membrane tension and the pressure generated by actin polymerization are two antagonistic forces believed to define the protrusion rate at the leading edge of migrating cells [1, 2, 3, 4 and 5]. Quantitatively, resistance to actin protrusion is a product of membrane tension and mean local curvature (Laplace’s law); thus, it depends on the local geometry of the membrane interface. However, the role of the geometry of the leading edge in protrusion control has not been yet investigated. Here, we manipulate both the cell shape and substrate topography in the model system of persistently migrating fish epidermal keratocytes. We find that the protrusion rate does not correlate with membrane tension, but, instead, strongly correlates with cell roundness, and that the leading edge of the cell exhibits pinning on substrate ridges—a phenomenon characteristic of spreading of liquid drops. These results indicate that the leading edge could be considered a triple interface between the substrate, membrane, and extracellular medium and that the contact angle between the membrane and the substrate determines the load on actin polymerization and, therefore, the protrusion rate. Our findings thus illuminate a novel relationship between the 3D shape of the cell and its dynamics, which may have implications for cell migration in 3D environments.
DOI
Potential energy surfaces and reaction pathways for light-mediated self-organization of metal nanoparticle clusters
Zijie Yan, Stephen K. Gray & Norbert F. Scherer
Potential energy surfaces are the central concept in understanding the assembly of molecules; atoms form molecules via covalent bonds with structures defined by the stationary points of the surfaces. Similarly, dispersion interactions give Lennard-Jones potentials that describe atomic clusters and liquids. The formation of molecules and clusters can follow various pathways depending on the initial conditions and the potentials. Here we show that analogous mechanistic effects occur in light-mediated self-organization of metal nanoparticles; atoms are replaced by silver nanoparticles that are arranged by electrodynamic (that is, optical trapping and optical binding) interactions. We demonstrate this concept using simple Gaussian optical fields and the formation of stable clusters with various two-dimensional (2D) and three-dimensional (3D) geometries. The formation of specific clusters is ‘path-dependent’; the particle motions follow an electrodynamic potential energy surface. This work paves the way for rational design of photonic clusters with combinations of imposed beam shapes, gradients and optical binding interactions.
DOI
Potential energy surfaces are the central concept in understanding the assembly of molecules; atoms form molecules via covalent bonds with structures defined by the stationary points of the surfaces. Similarly, dispersion interactions give Lennard-Jones potentials that describe atomic clusters and liquids. The formation of molecules and clusters can follow various pathways depending on the initial conditions and the potentials. Here we show that analogous mechanistic effects occur in light-mediated self-organization of metal nanoparticles; atoms are replaced by silver nanoparticles that are arranged by electrodynamic (that is, optical trapping and optical binding) interactions. We demonstrate this concept using simple Gaussian optical fields and the formation of stable clusters with various two-dimensional (2D) and three-dimensional (3D) geometries. The formation of specific clusters is ‘path-dependent’; the particle motions follow an electrodynamic potential energy surface. This work paves the way for rational design of photonic clusters with combinations of imposed beam shapes, gradients and optical binding interactions.
DOI
Wednesday, May 28, 2014
Thermodynamics: Engines and demons
Jörn Dunkel
Brownian motion in a feedback-controlled optical trap provides a minimal experimental realization of a Szilárd engine, confirming fluctuation theorems and demonstrating the importance of spontaneous symmetry breaking in small thermodynamic systems.
DOI
Brownian motion in a feedback-controlled optical trap provides a minimal experimental realization of a Szilárd engine, confirming fluctuation theorems and demonstrating the importance of spontaneous symmetry breaking in small thermodynamic systems.
DOI
Structured attachment of bacterial molecular motors for defined microflow induction
Mike Woerdemann, Florian Hörner, Cornelia Denz
Bacterial rotational motor complexes that propel flagellated bacteria possess unique properties like their size of a few nanometres and the ability of selfreproduction that have led to various exciting applications including biohybrid nano-machines. One mandatory prerequisite to utilize bacterial nano motors in fluid applications is the ability to transfer force and torque to the fluid, which usually can be achieved by attachment of the bacterial cell to adequate surfaces. Additionally, for optimal transfer of force or torque, precise control of the position down to the single cell level is of utmost importance. Based on a PIV (particle image velocimetry) evaluation of the induced flow of single bacteria,we propose and demonstrate attachment of arbitrary patterns of motile bacterial cells in a fast light-based two-step process for the first time to our knowledge. First, these cells are pre-structured by holographic optical tweezers and then attached to a homogeneous, polystyrene-coated surface. In contrast to the few approaches that have been implemented up to now and which rely on pre-structured surfaces, our scheme allows for precise control on a single bacterium level, is versatile, interactive and has low requirements with respect to the surface preparation.
DOI
Bacterial rotational motor complexes that propel flagellated bacteria possess unique properties like their size of a few nanometres and the ability of selfreproduction that have led to various exciting applications including biohybrid nano-machines. One mandatory prerequisite to utilize bacterial nano motors in fluid applications is the ability to transfer force and torque to the fluid, which usually can be achieved by attachment of the bacterial cell to adequate surfaces. Additionally, for optimal transfer of force or torque, precise control of the position down to the single cell level is of utmost importance. Based on a PIV (particle image velocimetry) evaluation of the induced flow of single bacteria,we propose and demonstrate attachment of arbitrary patterns of motile bacterial cells in a fast light-based two-step process for the first time to our knowledge. First, these cells are pre-structured by holographic optical tweezers and then attached to a homogeneous, polystyrene-coated surface. In contrast to the few approaches that have been implemented up to now and which rely on pre-structured surfaces, our scheme allows for precise control on a single bacterium level, is versatile, interactive and has low requirements with respect to the surface preparation.
DOI
Wavelength-dependent longitudinal polarizability of gold nanorod on optical torques
Jiunn-Woei Liaw, Wei-Jiun Lo, and Mao-Kuen Kuo
This study theoretically investigates the wavelength-dependent longitudinal polarizability of a gold nanorod (GNR) irradiated by a polarized laser beam. The resultant optical torque in terms of the Maxwell stress tensor was analyzed quantitatively using the multiple multipole method. Our results indicate that the real part of the longitudinal polarizability of GNR can be either positive or negative, leading to the parallel or perpendicular modes, respectively. For the parallel and perpendicular modes, the long axis of GNR is rotated to align parallel and perpendicular, respectively, to the polarization direction of the illuminating light. The turning point between these two modes, depending on the aspect ratio (AR) and the size of GNR, nearly coincides with the longitudinal surface plasmon resonance (LSPR). The perpendicular mode ranges from the transverse SPR to LSPR, and the range of the parallel mode is broadband from LSPR to the near infrared regime. Owing to that a larger optical torque and less plasmonic heating are of concern, an efficiency of optical torque is defined to evaluate the performance of different wavelengths. Analysis results indicate that lasers with wavelength in the perpendicular mode are applicable to rotate and align a GNR of a higher AR. For example, the laser of 785 nm (the perpendicular mode) is superior to that of 1064 nm (the parallel mode, off-resonant from LSPR of 955 nm) for rotating a GNR of AR = 4 and radius 20 nm with an orientation of 45° with respect to the laser polarization.
DOI
This study theoretically investigates the wavelength-dependent longitudinal polarizability of a gold nanorod (GNR) irradiated by a polarized laser beam. The resultant optical torque in terms of the Maxwell stress tensor was analyzed quantitatively using the multiple multipole method. Our results indicate that the real part of the longitudinal polarizability of GNR can be either positive or negative, leading to the parallel or perpendicular modes, respectively. For the parallel and perpendicular modes, the long axis of GNR is rotated to align parallel and perpendicular, respectively, to the polarization direction of the illuminating light. The turning point between these two modes, depending on the aspect ratio (AR) and the size of GNR, nearly coincides with the longitudinal surface plasmon resonance (LSPR). The perpendicular mode ranges from the transverse SPR to LSPR, and the range of the parallel mode is broadband from LSPR to the near infrared regime. Owing to that a larger optical torque and less plasmonic heating are of concern, an efficiency of optical torque is defined to evaluate the performance of different wavelengths. Analysis results indicate that lasers with wavelength in the perpendicular mode are applicable to rotate and align a GNR of a higher AR. For example, the laser of 785 nm (the perpendicular mode) is superior to that of 1064 nm (the parallel mode, off-resonant from LSPR of 955 nm) for rotating a GNR of AR = 4 and radius 20 nm with an orientation of 45° with respect to the laser polarization.
DOI
Transcription factors IIS and IIF enhance transcription efficiency by differentially modifying RNA polymerase pausing dynamics
Toyotaka Ishibashi, Manchuta Dangkulwanich, Yves Coello,Troy A. Lionberger, Lucyna Lubkowska, Alfred S. Ponticelli, Mikhail Kashlev, and Carlos Bustamante
Transcription factors IIS (TFIIS) and IIF (TFIIF) are known to stimulate transcription elongation. Here, we use a single-molecule transcription elongation assay to study the effects of both factors. We find that these transcription factors enhance overall transcription elongation by reducing the lifetime of transcriptional pauses and that TFIIF also decreases the probability of pause entry. Furthermore, we observe that both factors enhance the processivity of RNA polymerase II through the nucleosomal barrier. The effects of TFIIS and TFIIF are quantitatively described using the linear Brownian ratchet kinetic model for transcription elongation and the backtracking model for transcriptional pauses, modified to account for the effects of the transcription factors. Our findings help elucidate the molecular mechanisms by which transcription factors modulate gene expression.
Transcription factors IIS (TFIIS) and IIF (TFIIF) are known to stimulate transcription elongation. Here, we use a single-molecule transcription elongation assay to study the effects of both factors. We find that these transcription factors enhance overall transcription elongation by reducing the lifetime of transcriptional pauses and that TFIIF also decreases the probability of pause entry. Furthermore, we observe that both factors enhance the processivity of RNA polymerase II through the nucleosomal barrier. The effects of TFIIS and TFIIF are quantitatively described using the linear Brownian ratchet kinetic model for transcription elongation and the backtracking model for transcriptional pauses, modified to account for the effects of the transcription factors. Our findings help elucidate the molecular mechanisms by which transcription factors modulate gene expression.
Weak Electrolyte Dependence in the Repulsion of Colloids at an Oil–Water Interface
Christopher L. Wirth, Eric M. Furst, and Jan Vermant
The two-particle interaction between 3.1-μm-diameter polystyrene latex particles at a decane–water interface was measured with time-shared optical tweezers. The water subphase contained either 0.103 mM RbCl or 0.0342 mM MgCl2, which have hydrated cations of different size but identical anions. The choice of both the anion and the concentrations makes a comparison with published data on NaCl possible and also isolates the effect of the nature of the cation on the electrostatic interaction. The measured magnitude of the dipolar force and the relative changes as a function of electrolyte were in quantitative agreement with predictions from a recently published model that uses the Langevin–Poisson–Boltzmann equation including steric effects and the polarization saturation of the medium to predict the dipolar interaction (Frydel, D.; Oettel, M. Phys. Chem. Chem. Phys. 2011, 13, 4109–4118). These results support the hypothesis that a condensed layer of counterions contributes to the electrostatic interaction between colloidal particles at an oil–water interface. Although it has been suggested that the electrostatic interactions between particles at liquid interfaces could serve as a sensitive probe of the structural details of the electric double layer, both the model predictions and experimental measurements showed a maximum change of only 25% in the magnitude of the interaction with a change in electrolyte under the conditions tested. The ability to resolve this small change was confounded by the heterogeneous nature of the interaction. Thus, despite the apparent importance of the choice of electrolyte, the subtlety of competing effects makes it unlikely that colloidal force measurements could be used to probe the fine structure of the electric double layer.
DOI
The two-particle interaction between 3.1-μm-diameter polystyrene latex particles at a decane–water interface was measured with time-shared optical tweezers. The water subphase contained either 0.103 mM RbCl or 0.0342 mM MgCl2, which have hydrated cations of different size but identical anions. The choice of both the anion and the concentrations makes a comparison with published data on NaCl possible and also isolates the effect of the nature of the cation on the electrostatic interaction. The measured magnitude of the dipolar force and the relative changes as a function of electrolyte were in quantitative agreement with predictions from a recently published model that uses the Langevin–Poisson–Boltzmann equation including steric effects and the polarization saturation of the medium to predict the dipolar interaction (Frydel, D.; Oettel, M. Phys. Chem. Chem. Phys. 2011, 13, 4109–4118). These results support the hypothesis that a condensed layer of counterions contributes to the electrostatic interaction between colloidal particles at an oil–water interface. Although it has been suggested that the electrostatic interactions between particles at liquid interfaces could serve as a sensitive probe of the structural details of the electric double layer, both the model predictions and experimental measurements showed a maximum change of only 25% in the magnitude of the interaction with a change in electrolyte under the conditions tested. The ability to resolve this small change was confounded by the heterogeneous nature of the interaction. Thus, despite the apparent importance of the choice of electrolyte, the subtlety of competing effects makes it unlikely that colloidal force measurements could be used to probe the fine structure of the electric double layer.
DOI
Saturday, May 24, 2014
Optical trapping and Raman spectroscopy of solid particles
L. Rkiouak, M. J. Tang, J. C. J. Camp, J. McGregor, I. M. Watson, R. A. Cox, M. Kalberer, A. D. Ward and F. D. Pope
The heterogeneous interactions of gas molecules on solid particles are crucial in many areas of science, engineering and technology. Such interactions play a critical role in atmospheric chemistry and in heterogeneous catalysis, a key technology in the energy and chemical industries. Investigating heterogeneous interactions upon single levitated particles can provide significant insight into these important processes. Various methodologies exist for levitating micron sized particles including: optical, electrical and acoustic techniques. Prior to this study, the optical levitation of solid micron scale particles has proved difficult to achieve over timescales relevant to the above applications. In this work, a new vertically configured counter propagating dual beam optical trap was optimized to levitate a range of solid particles in air. Silica (SiO2), α-alumina (Al2O3), titania (TiO2) and polystyrene were stably trapped with a high trapping efficiency (Q = 0.42). The longest stable trapping experiment was conducted continuously for 24 hours, and there are no obvious constraints on trapping time beyond this period. Therefore, the methodology described in this paper should be of major benefit to various research communities. The strength of the new technique is demonstrated by the simultaneous levitation and spectroscopic interrogation of silica particles by Raman spectroscopy. In particular, the adsorption of water upon silica was investigated under controlled relative humidity environments. Furthermore, the collision and coagulation behaviour of silica particles with microdroplets of sulphuric acid was followed using both optical imaging and Raman spectroscopy.
DOI
The heterogeneous interactions of gas molecules on solid particles are crucial in many areas of science, engineering and technology. Such interactions play a critical role in atmospheric chemistry and in heterogeneous catalysis, a key technology in the energy and chemical industries. Investigating heterogeneous interactions upon single levitated particles can provide significant insight into these important processes. Various methodologies exist for levitating micron sized particles including: optical, electrical and acoustic techniques. Prior to this study, the optical levitation of solid micron scale particles has proved difficult to achieve over timescales relevant to the above applications. In this work, a new vertically configured counter propagating dual beam optical trap was optimized to levitate a range of solid particles in air. Silica (SiO2), α-alumina (Al2O3), titania (TiO2) and polystyrene were stably trapped with a high trapping efficiency (Q = 0.42). The longest stable trapping experiment was conducted continuously for 24 hours, and there are no obvious constraints on trapping time beyond this period. Therefore, the methodology described in this paper should be of major benefit to various research communities. The strength of the new technique is demonstrated by the simultaneous levitation and spectroscopic interrogation of silica particles by Raman spectroscopy. In particular, the adsorption of water upon silica was investigated under controlled relative humidity environments. Furthermore, the collision and coagulation behaviour of silica particles with microdroplets of sulphuric acid was followed using both optical imaging and Raman spectroscopy.
DOI
Nanophotonic trapping for precise manipulation of biomolecular arrays
Mohammad Soltani, Jun Lin, Robert A. Forties, James T. Inman, Summer N. Saraf, Robert M. Fulbright, Michal Lipson & Michelle D. Wang
Optical trapping is a powerful manipulation and measurement technique widely used in the biological and materials sciences. Miniaturizing optical trap instruments onto optofluidic platforms holds promise for high-throughput lab-on-a-chip applications. However, a persistent challenge with existing optofluidic devices has been achieving controlled and precise manipulation of trapped particles. Here, we report a new class of on-chip optical trapping devices. Using photonic interference functionalities, an array of stable, three-dimensional on-chip optical traps is formed at the antinodes of a standing-wave evanescent field on a nanophotonic waveguide. By employing the thermo-optic effect via integrated electric microheaters, the traps can be repositioned at high speed (~30 kHz) with nanometre precision. We demonstrate sorting and manipulation of individual DNA molecules. In conjunction with laminar flows and fluorescence, we also show precise control of the chemical environment of a sample with simultaneous monitoring. Such a controllable trapping device has the potential to achieve high-throughput precision measurements on chip.
DOI
Optical trapping is a powerful manipulation and measurement technique widely used in the biological and materials sciences. Miniaturizing optical trap instruments onto optofluidic platforms holds promise for high-throughput lab-on-a-chip applications. However, a persistent challenge with existing optofluidic devices has been achieving controlled and precise manipulation of trapped particles. Here, we report a new class of on-chip optical trapping devices. Using photonic interference functionalities, an array of stable, three-dimensional on-chip optical traps is formed at the antinodes of a standing-wave evanescent field on a nanophotonic waveguide. By employing the thermo-optic effect via integrated electric microheaters, the traps can be repositioned at high speed (~30 kHz) with nanometre precision. We demonstrate sorting and manipulation of individual DNA molecules. In conjunction with laminar flows and fluorescence, we also show precise control of the chemical environment of a sample with simultaneous monitoring. Such a controllable trapping device has the potential to achieve high-throughput precision measurements on chip.
DOI
Experimental observation of particle cones formed by optical trapping
Chuji Wang, Yong-Le Pan, and Mark Coleman
We report on observation of particle cones formed by optical trapping of absorbing particles in air using two sets of simple geometric optical schemes. Further, the trapped particles on a cone in both schemes are size-sorted with large particles or particle ensembles close to the cone vertex. This new experimental observation shows an excellent example of 3D particle trapping between the two extreme cases, photon radiation trapping of nonabsorbing particles and photophoretic trapping of strongly absorbing particles; and the observation may challenge theoretical calculations of the trapping forces applied in this case.
DOI
We report on observation of particle cones formed by optical trapping of absorbing particles in air using two sets of simple geometric optical schemes. Further, the trapped particles on a cone in both schemes are size-sorted with large particles or particle ensembles close to the cone vertex. This new experimental observation shows an excellent example of 3D particle trapping between the two extreme cases, photon radiation trapping of nonabsorbing particles and photophoretic trapping of strongly absorbing particles; and the observation may challenge theoretical calculations of the trapping forces applied in this case.
DOI
Force Unfolding Kinetics of RNA using Optical Tweezers. II. Modeling Experiments
M. Manosas, J.-D. Wen, P.T.X. Li, S.B. Smith, C. Bustamante, I. Tinoco Jr., F. Ritort
By exerting mechanical force, it is possible to unfold/refold RNA molecules one at a time. In a small range of forces, an RNA molecule can hop between the folded and the unfolded state with force-dependent kinetic rates. Here, we introduce a mesoscopic model to analyze the hopping kinetics of RNA hairpins in an optical tweezers setup. The model includes different elements of the experimental setup (beads, handles, and RNA sequence) and limitations of the instrument (time lag of the force-feedback mechanism and finite bandwidth of data acquisition). We investigated the influence of the instrument on the measured hopping rates. Results from the model are in good agreement with the experiments reported in the companion article. The comparison between theory and experiments allowed us to infer the values of the intrinsic molecular rates of the RNA hairpin alone and to search for the optimal experimental conditions to do the measurements. We conclude that the longest handles and softest traps that allow detection of the folding/unfolding signal (handles ∼5–10 Kbp and traps ∼0.03 pN/nm) represent the best conditions to obtain the intrinsic molecular rates. The methodology and rationale presented here can be applied to other experimental setups and other molecules.
DOI
By exerting mechanical force, it is possible to unfold/refold RNA molecules one at a time. In a small range of forces, an RNA molecule can hop between the folded and the unfolded state with force-dependent kinetic rates. Here, we introduce a mesoscopic model to analyze the hopping kinetics of RNA hairpins in an optical tweezers setup. The model includes different elements of the experimental setup (beads, handles, and RNA sequence) and limitations of the instrument (time lag of the force-feedback mechanism and finite bandwidth of data acquisition). We investigated the influence of the instrument on the measured hopping rates. Results from the model are in good agreement with the experiments reported in the companion article. The comparison between theory and experiments allowed us to infer the values of the intrinsic molecular rates of the RNA hairpin alone and to search for the optimal experimental conditions to do the measurements. We conclude that the longest handles and softest traps that allow detection of the folding/unfolding signal (handles ∼5–10 Kbp and traps ∼0.03 pN/nm) represent the best conditions to obtain the intrinsic molecular rates. The methodology and rationale presented here can be applied to other experimental setups and other molecules.
DOI
Force Unfolding Kinetics of RNA Using Optical Tweezers. I. Effects of Experimental Variables on Measured Results
Jin-Der Wen, Maria Manosas, Pan T.X. Li, Steven B. Smith, Carlos Bustamante, Felix Ritort, Ignacio Tinoco Jr.
Experimental variables of optical tweezers instrumentation that affect RNA folding/unfolding kinetics were investigated. A model RNA hairpin, P5ab, was attached to two micron-sized beads through hybrid RNA/DNA handles; one bead was trapped by dual-beam lasers and the other was held by a micropipette. Several experimental variables were changed while measuring the unfolding/refolding kinetics, including handle lengths, trap stiffness, and modes of force applied to the molecule. In constant-force mode where the tension applied to the RNA was maintained through feedback control, the measured rate coefficients varied within 40% when the handle lengths were changed by 10-fold (1.1–10.2 Kbp); they increased by two- to threefold when the trap stiffness was lowered to one-third (from 0.1 to 0.035 pN/nm). In the passive mode, without feedback control and where the force applied to the RNA varied in response to the end-to-end distance change of the tether, the RNA hopped between a high-force folded-state and a low-force unfolded-state. In this mode, the rates increased up to twofold with longer handles or softer traps. Overall, the measured rates remained with the same order-of-magnitude over the wide range of conditions studied. In the companion article on pages 3010–3021, we analyze how the measured kinetics parameters differ from the intrinsic molecular rates of the RNA, and thus how to obtain the molecular rates.
DOI
Experimental variables of optical tweezers instrumentation that affect RNA folding/unfolding kinetics were investigated. A model RNA hairpin, P5ab, was attached to two micron-sized beads through hybrid RNA/DNA handles; one bead was trapped by dual-beam lasers and the other was held by a micropipette. Several experimental variables were changed while measuring the unfolding/refolding kinetics, including handle lengths, trap stiffness, and modes of force applied to the molecule. In constant-force mode where the tension applied to the RNA was maintained through feedback control, the measured rate coefficients varied within 40% when the handle lengths were changed by 10-fold (1.1–10.2 Kbp); they increased by two- to threefold when the trap stiffness was lowered to one-third (from 0.1 to 0.035 pN/nm). In the passive mode, without feedback control and where the force applied to the RNA varied in response to the end-to-end distance change of the tether, the RNA hopped between a high-force folded-state and a low-force unfolded-state. In this mode, the rates increased up to twofold with longer handles or softer traps. Overall, the measured rates remained with the same order-of-magnitude over the wide range of conditions studied. In the companion article on pages 3010–3021, we analyze how the measured kinetics parameters differ from the intrinsic molecular rates of the RNA, and thus how to obtain the molecular rates.
DOI
Thursday, May 22, 2014
Mutations in BIN1 Associated with Centronuclear Myopathy Disrupt Membrane Remodeling by Affecting Protein Density and Oligomerization
Tingting Wu, Zheng Shi, Tobias Baumgart
The regulation of membrane shapes is central to many cellular phenomena. Bin/Amphiphysin/Rvs (BAR) domain-containing proteins are key players for membrane remodeling during endocytosis, cell migration, and endosomal sorting. BIN1, which contains an N-BAR domain, is assumed to be essential for biogenesis of plasma membrane invaginations (T-tubules) in muscle tissues. Three mutations, K35N, D151N and R154Q, have been discovered so far in the BAR domain of BIN1 in patients with centronuclear myopathy (CNM), where impaired organization of T-tubules has been reported. However, molecular mechanisms behind this malfunction have remained elusive. None of the BIN1 disease mutants displayed a significantly compromised curvature sensing ability. However, two mutants showed impaired membrane tubulation both in vivo and in vitro, and displayed characteristically different behaviors. R154Q generated smaller membrane curvature compared to WT N-BAR. Quantification of protein density on membranes revealed a lower membrane-bound density for R154Q compared to WT and the other mutants, which appeared to be the primary reason for the observation of impaired deformation capacity. The D151N mutant was unable to tubulate liposomes under certain experimental conditions. At medium protein concentrations we found ‘budding’ structures on liposomes that we hypothesized to be intermediates during the tubulation process except for the D151N mutant. Chemical crosslinking assays suggested that the D151N mutation impaired protein oligomerization upon membrane binding. Although we found an insignificant difference between WT and K35N N-BAR in in vitro assays, depolymerizing actin in live cells allowed tubulation of plasma membranes through the K35N mutant. Our results provide insights into the membrane-involved pathophysiological mechanisms leading to human disease.
DOI
The regulation of membrane shapes is central to many cellular phenomena. Bin/Amphiphysin/Rvs (BAR) domain-containing proteins are key players for membrane remodeling during endocytosis, cell migration, and endosomal sorting. BIN1, which contains an N-BAR domain, is assumed to be essential for biogenesis of plasma membrane invaginations (T-tubules) in muscle tissues. Three mutations, K35N, D151N and R154Q, have been discovered so far in the BAR domain of BIN1 in patients with centronuclear myopathy (CNM), where impaired organization of T-tubules has been reported. However, molecular mechanisms behind this malfunction have remained elusive. None of the BIN1 disease mutants displayed a significantly compromised curvature sensing ability. However, two mutants showed impaired membrane tubulation both in vivo and in vitro, and displayed characteristically different behaviors. R154Q generated smaller membrane curvature compared to WT N-BAR. Quantification of protein density on membranes revealed a lower membrane-bound density for R154Q compared to WT and the other mutants, which appeared to be the primary reason for the observation of impaired deformation capacity. The D151N mutant was unable to tubulate liposomes under certain experimental conditions. At medium protein concentrations we found ‘budding’ structures on liposomes that we hypothesized to be intermediates during the tubulation process except for the D151N mutant. Chemical crosslinking assays suggested that the D151N mutation impaired protein oligomerization upon membrane binding. Although we found an insignificant difference between WT and K35N N-BAR in in vitro assays, depolymerizing actin in live cells allowed tubulation of plasma membranes through the K35N mutant. Our results provide insights into the membrane-involved pathophysiological mechanisms leading to human disease.
DOI
Measurements of the force fields within an acoustic standing wave using holographic optical tweezer
P. G. Bassindale, D. B. Phillips, A. C. Barnes and B. W. Drinkwater
Direct measurement of the forces experienced by micro-spheres in an acoustic standing wave device have been obtained using calibrated optical traps generated with holographic optical tweezers. A micro-sphere, which is optically trapped in three dimensions, can be moved through the acoustic device to measure forces acting upon it. When the micro-sphere is subjected to acoustic forces, it's equilibrium position is displaced to a position where the acoustic forces and optical forces are balanced. Once the optical trapping stiffness has been calibrated, observation of this displacement enables a direct measurement of the forces acting upon the micro-sphere. The measured forces are separated into a spatially oscillating component, attributed to the acoustic radiation force, and a constant force, attributed to fluid streaming. As the drive conditions of the acoustic device were varied, oscillating forces (>2.5 pN pp ) and streaming forces (<0.2 pN) were measured. A 5 μm silica micro-sphere was used to characterise a 6.8 MHz standing wave, λ = 220 μm, to a spatial resolution limited by the uncertainty in the positioning of the micro-sphere (here to within 2 nm) and with a force resolution on the order of 10 fN. The results have application in the design and testing of acoustic manipulation devices.
DOI
Direct measurement of the forces experienced by micro-spheres in an acoustic standing wave device have been obtained using calibrated optical traps generated with holographic optical tweezers. A micro-sphere, which is optically trapped in three dimensions, can be moved through the acoustic device to measure forces acting upon it. When the micro-sphere is subjected to acoustic forces, it's equilibrium position is displaced to a position where the acoustic forces and optical forces are balanced. Once the optical trapping stiffness has been calibrated, observation of this displacement enables a direct measurement of the forces acting upon the micro-sphere. The measured forces are separated into a spatially oscillating component, attributed to the acoustic radiation force, and a constant force, attributed to fluid streaming. As the drive conditions of the acoustic device were varied, oscillating forces (>2.5 pN pp ) and streaming forces (<0.2 pN) were measured. A 5 μm silica micro-sphere was used to characterise a 6.8 MHz standing wave, λ = 220 μm, to a spatial resolution limited by the uncertainty in the positioning of the micro-sphere (here to within 2 nm) and with a force resolution on the order of 10 fN. The results have application in the design and testing of acoustic manipulation devices.
DOI
Scattering approach to two-colour light forces and self-ordering of polarizable particles
S Ostermann, M Sonnleitner and H Ritsch
Collective coherent scattering of laser light by an ensemble of polarizable point particles creates long-range interactions, whose properties can be tailored by the choice of injected laser powers, frequencies, and polarizations. We use a transfer matrix approach to study the forces induced by non-interfering fields of orthogonal polarization or different frequencies in a 1D geometry, and find long-range self-ordering of particles without a prescribed order. Adjusting the laser frequencies and powers allows one to tune the inter-particle distances and provides a wide range of possible dynamical couplings not accessible in usual standing wave geometries with prescribed order. In this work, we restrict the examples to two frequencies and polarizations, but the framework also allows one to treat multicolour light beams with random phases. These dynamical effects should be observable in existing experimental setups with effective 1D geometries, such as atoms or nanoparticles coupled to the field of an optical nanofibre or transversely trapped in counter-propagating Gaussian beams.
DOI
Collective coherent scattering of laser light by an ensemble of polarizable point particles creates long-range interactions, whose properties can be tailored by the choice of injected laser powers, frequencies, and polarizations. We use a transfer matrix approach to study the forces induced by non-interfering fields of orthogonal polarization or different frequencies in a 1D geometry, and find long-range self-ordering of particles without a prescribed order. Adjusting the laser frequencies and powers allows one to tune the inter-particle distances and provides a wide range of possible dynamical couplings not accessible in usual standing wave geometries with prescribed order. In this work, we restrict the examples to two frequencies and polarizations, but the framework also allows one to treat multicolour light beams with random phases. These dynamical effects should be observable in existing experimental setups with effective 1D geometries, such as atoms or nanoparticles coupled to the field of an optical nanofibre or transversely trapped in counter-propagating Gaussian beams.
DOI
Saturday, May 17, 2014
Enhancing optofluidic actuation of micro-objects by tagging with plasmonic nanoparticles
Julien Burgin, Satyabrata Si, Marie-Hélène Delville, and Jean-Pierre Delville
We report experimentally and theoretically on the significant exaltation of optical forces on microparticles when they are partially coated by metallic nanodots and shined with laser light within the surface plasmon resonance. Optical forces on both pure silica particles and silica-gold raspberries are characterized using an optical chromatography setup to measure the variations of the Stokes drag versus laser beam power. Results are compared to the Mie theory prediction for both pure dielectric particles and core-shell ones with a shell described as a continuous dielectric-metal composite of dielectric constant determined from the Maxwell-Garnett approach. The observed quantitative agreement demonstrates that radiation pressure forces are directly related to the metal concentration on the microparticle surface and that metallic nanodots increase the magnitude of optical forces compared to pure dielectric particles of the same overall size, even at very low metal concentration. Behaving as “micro-sized nanoparticles”, the benefit of microparticles coated with metallic nanodots is thus twofold: it significantly enhances optofluidic manipulation and motion at the microscale, and brings nanometric optical, chemical or biological capabilities to the microscale.
DOI
We report experimentally and theoretically on the significant exaltation of optical forces on microparticles when they are partially coated by metallic nanodots and shined with laser light within the surface plasmon resonance. Optical forces on both pure silica particles and silica-gold raspberries are characterized using an optical chromatography setup to measure the variations of the Stokes drag versus laser beam power. Results are compared to the Mie theory prediction for both pure dielectric particles and core-shell ones with a shell described as a continuous dielectric-metal composite of dielectric constant determined from the Maxwell-Garnett approach. The observed quantitative agreement demonstrates that radiation pressure forces are directly related to the metal concentration on the microparticle surface and that metallic nanodots increase the magnitude of optical forces compared to pure dielectric particles of the same overall size, even at very low metal concentration. Behaving as “micro-sized nanoparticles”, the benefit of microparticles coated with metallic nanodots is thus twofold: it significantly enhances optofluidic manipulation and motion at the microscale, and brings nanometric optical, chemical or biological capabilities to the microscale.
DOI
High-frequency affine mechanics and nonaffine relaxation in a model cytoskeleton
David A. Head, Emi Ikebe, Akiko Nakamasu, Peijuan Zhang, Lara Gay Villaruz, Suguru Kinoshita, Shoji Ando, and Daisuke Mizuno
The cytoskeleton is a network of crosslinked, semiflexible filaments, and it has been suggested that it has properties of a glassy state. Here we employ optical-trap-based microrheology to apply forces to a model cytoskeleton and measure the high-bandwidth response at an anterior point. Simulating the highly nonlinear and anisotropic stress-strain propagation assuming affinity, we found that theoretical predictions for the quasistatic response of semiflexible polymers are only realized at high frequencies inaccessible to conventional rheometers. We give a theoretical basis for determining the frequency when both affinity and quasistaticity are valid, and we discuss with experimental evidence that the relaxations at lower frequencies can be characterized by the experimentally obtained nonaffinity parameter.
DOI
The cytoskeleton is a network of crosslinked, semiflexible filaments, and it has been suggested that it has properties of a glassy state. Here we employ optical-trap-based microrheology to apply forces to a model cytoskeleton and measure the high-bandwidth response at an anterior point. Simulating the highly nonlinear and anisotropic stress-strain propagation assuming affinity, we found that theoretical predictions for the quasistatic response of semiflexible polymers are only realized at high frequencies inaccessible to conventional rheometers. We give a theoretical basis for determining the frequency when both affinity and quasistaticity are valid, and we discuss with experimental evidence that the relaxations at lower frequencies can be characterized by the experimentally obtained nonaffinity parameter.
DOI
Propagation dynamics and optical trapping of a radial Airy array beam
Ke Cheng, Xianqiong Zhong, Anping Xiang
Analytical propagation expression of a radial Airy array beam in coherent and incoherent combination passing through paraxial ABCD system is derived, and used to investigate the effect of combination scheme, array orientation and initial phase of Airy beamlet on propagation dynamics of the resulting beam in free space, where optical spot array and vortex array with different shapes are also found, respectively. And then taking four-beamlet Airy array beam in same array orientation as an example, square optical spot array obtained in focal field can be used for simultaneous trapping multiple Rayleigh particles with relative refractive index larger than 1. The transverse gradient forces serving as restore forces tend to push particles at different initial positions to their individual optical spot center. The analysis of trapping stability indicates that larger input peak intensity of Airy beamlet and smaller particle size are benefit to trapping particle owing to many deeper potential wells. Vortex array produced by coherent combined Airy array beam in this paper is expected to be useful for simultaneous trapping microparticles with relative refractive index smaller than 1.
DOI
Analytical propagation expression of a radial Airy array beam in coherent and incoherent combination passing through paraxial ABCD system is derived, and used to investigate the effect of combination scheme, array orientation and initial phase of Airy beamlet on propagation dynamics of the resulting beam in free space, where optical spot array and vortex array with different shapes are also found, respectively. And then taking four-beamlet Airy array beam in same array orientation as an example, square optical spot array obtained in focal field can be used for simultaneous trapping multiple Rayleigh particles with relative refractive index larger than 1. The transverse gradient forces serving as restore forces tend to push particles at different initial positions to their individual optical spot center. The analysis of trapping stability indicates that larger input peak intensity of Airy beamlet and smaller particle size are benefit to trapping particle owing to many deeper potential wells. Vortex array produced by coherent combined Airy array beam in this paper is expected to be useful for simultaneous trapping microparticles with relative refractive index smaller than 1.
DOI
Friday, May 16, 2014
Transverse spinning of a sphere in a plasmonic field
Antoine Canaguier-Durand and Cyriaque Genet
We evaluate optical forces and torques induced by a surface plasmon to a sphere of arbitrary size, i.e., beyond the pointlike dipolar limit. Through a multipolar decomposition of the plasmonic field, we demonstrate that the induced torque is purely transverse to the plasmon propagation direction. Our approach removes the inherent ambiguities of the dipolar regime with respect to rotations and emphasizes the crucial role played by dissipation in the onset of the plasmonic torque. We also give realistic estimates of such plasmon-induced spinning of gold spheres immersed in water or air.
DOI
We evaluate optical forces and torques induced by a surface plasmon to a sphere of arbitrary size, i.e., beyond the pointlike dipolar limit. Through a multipolar decomposition of the plasmonic field, we demonstrate that the induced torque is purely transverse to the plasmon propagation direction. Our approach removes the inherent ambiguities of the dipolar regime with respect to rotations and emphasizes the crucial role played by dissipation in the onset of the plasmonic torque. We also give realistic estimates of such plasmon-induced spinning of gold spheres immersed in water or air.
DOI
Nonlinear optomechanical pressure
Claudio Conti and Robert Boyd
A transparent material exhibits ultrafast optical nonlinearity and is subject to optical pressure if irradiated by a laser beam. However, the effect of nonlinearity on optical pressure is often overlooked, even if a nonlinear optical pressure may be potentially employed in many applications, such as optical manipulation, biophysics, cavity optomechanics, quantum optics, and optical tractors, and is relevant in fundamental problems such as the Abraham-Minkoswky dilemma or the Casimir effect. Here, we show that an ultrafast nonlinear polarization gives indeed a contribution to the optical pressure that also is negative in certain spectral ranges; the theoretical analysis is confirmed by first-principles simulations. An order-of-magnitude estimate shows that the effect can be observable by measuring the deflection of a membrane made by graphene.
DOI
A transparent material exhibits ultrafast optical nonlinearity and is subject to optical pressure if irradiated by a laser beam. However, the effect of nonlinearity on optical pressure is often overlooked, even if a nonlinear optical pressure may be potentially employed in many applications, such as optical manipulation, biophysics, cavity optomechanics, quantum optics, and optical tractors, and is relevant in fundamental problems such as the Abraham-Minkoswky dilemma or the Casimir effect. Here, we show that an ultrafast nonlinear polarization gives indeed a contribution to the optical pressure that also is negative in certain spectral ranges; the theoretical analysis is confirmed by first-principles simulations. An order-of-magnitude estimate shows that the effect can be observable by measuring the deflection of a membrane made by graphene.
DOI
Monday, May 12, 2014
Allosteric regulation of phosphofructokinase controls the emergence of glycolytic oscillations in isolated yeast cells
Anna-Karin Gustavsson, David D. van Niekerk, Caroline B. Adiels, Bob Kooi, Mattias Goksör and Jacky L. Snoep
Oscillations are widely distributed in nature and synchronization of oscillators has been described at the cellular level (e.g. heart cells) and at the population level (e.g. fireflies). Yeast glycolysis is the best known oscillatory system, although it has been studied almost exclusively at the population level (i.e. limited to observations of average behaviour in synchronized cultures). We studied individual yeast cells that were positioned with optical tweezers in a microfluidic chamber to determine the precise conditions for autonomous glycolytic oscillations. Hopf bifurcation points were determined experimentally in individual cells as a function of glucose and cyanide concentrations. The experiments were analyzed in a detailed mathematical model and could be interpreted in terms of an oscillatory manifold in a three-dimensional state-space; crossing the boundaries of the manifold coincides with the onset of oscillations and positioning along the longitudinal axis of the volume sets the period. The oscillatory manifold could be approximated by allosteric control values of phosphofructokinase for ATP and AMP.
DOI
Oscillations are widely distributed in nature and synchronization of oscillators has been described at the cellular level (e.g. heart cells) and at the population level (e.g. fireflies). Yeast glycolysis is the best known oscillatory system, although it has been studied almost exclusively at the population level (i.e. limited to observations of average behaviour in synchronized cultures). We studied individual yeast cells that were positioned with optical tweezers in a microfluidic chamber to determine the precise conditions for autonomous glycolytic oscillations. Hopf bifurcation points were determined experimentally in individual cells as a function of glucose and cyanide concentrations. The experiments were analyzed in a detailed mathematical model and could be interpreted in terms of an oscillatory manifold in a three-dimensional state-space; crossing the boundaries of the manifold coincides with the onset of oscillations and positioning along the longitudinal axis of the volume sets the period. The oscillatory manifold could be approximated by allosteric control values of phosphofructokinase for ATP and AMP.
DOI
Single-Molecule Force Spectroscopy of Rapidly Fluctuating, Marginally Stable Structures in the Intrinsically Disordered Protein α-Synuclein
Allison Solanki, Krishna Neupane, and Michael T. Woodside
Intrinsically disordered proteins form transient, fluctuating structures that are difficult to observe directly. We used optical tweezers to apply force to single α-synuclein molecules and measure their extension, characterizing the resulting conformational transitions. Force-extension curves revealed rapid fluctuations at low force, arising from the folding of two different classes of structure that were only marginally stable. The energy landscape for these transitions was characterized via the force-dependent kinetics derived from correlation analysis of the extension trajectories. The barriers were small, only a few kBT, but the diffusion was slow, revealing a landscape that is flat but rough.
DOI
Intrinsically disordered proteins form transient, fluctuating structures that are difficult to observe directly. We used optical tweezers to apply force to single α-synuclein molecules and measure their extension, characterizing the resulting conformational transitions. Force-extension curves revealed rapid fluctuations at low force, arising from the folding of two different classes of structure that were only marginally stable. The energy landscape for these transitions was characterized via the force-dependent kinetics derived from correlation analysis of the extension trajectories. The barriers were small, only a few kBT, but the diffusion was slow, revealing a landscape that is flat but rough.
DOI
Focusing properties of Gaussian beam with superimposed left-handed and right-handed helical phase fronts
Jinsong Li, Na Meng
Vector diffraction theory is employed to investigate the focusing properties of the Gaussian beams with superimposed left-handed and right-handed helical phase fronts theoretically. Numerical simulations show that the intensity distribution in focal region can be altered considerably by adjusting topological charge m corresponding to right-handed helix and topological charge n corresponding to left-handed helix. Many novel focal pattern may occur. It was shown that the focal pattern evolves from one intensity peak to multiple intensity peaks with changing the topological charge m and n, and all the intensity peaks form in a circle. As the number of intensity peaks is the sum of m and n, the focal pattern can be controlled through adjusting the topological charge m and n. And in order to show the possible applications of these alterable foci pattern, some optical gradient force distributions were also calculated and illuminated.
DOI
Vector diffraction theory is employed to investigate the focusing properties of the Gaussian beams with superimposed left-handed and right-handed helical phase fronts theoretically. Numerical simulations show that the intensity distribution in focal region can be altered considerably by adjusting topological charge m corresponding to right-handed helix and topological charge n corresponding to left-handed helix. Many novel focal pattern may occur. It was shown that the focal pattern evolves from one intensity peak to multiple intensity peaks with changing the topological charge m and n, and all the intensity peaks form in a circle. As the number of intensity peaks is the sum of m and n, the focal pattern can be controlled through adjusting the topological charge m and n. And in order to show the possible applications of these alterable foci pattern, some optical gradient force distributions were also calculated and illuminated.
DOI
Friday, May 9, 2014
Measurement of the microscopic viscosities of microfluids with a dynamic optical tweezers system
Yuquan Zhang, Xiaojing Wu, Yijia Wang, Siwei Zhu, Bruce Z Gao and X-C Yuan
Viscosity coefficients of microfluids—Newtonian and non-Newtonian—were explored through the rotational motion of a particle trapped by optical tweezers in a microflute. Unlike conventional methods based on viscometers, our microfluidic system employs samples of less than 30 μl to complete a measurement. Viscosity coefficients of ethanol and fetal bovine serum, as typical examples of Newtonian and non-Newtonian fluids, were obtained experimentally, and found to be in excellent agreement with theoretical predictions. Additionally, a practical application to a DNA solution with incremental ethidium bromide content was employed and the results are consistent with clinical data, indicating that our system provides a potentially important complementary tool for use in such biological and medical applications.
DOI
Viscosity coefficients of microfluids—Newtonian and non-Newtonian—were explored through the rotational motion of a particle trapped by optical tweezers in a microflute. Unlike conventional methods based on viscometers, our microfluidic system employs samples of less than 30 μl to complete a measurement. Viscosity coefficients of ethanol and fetal bovine serum, as typical examples of Newtonian and non-Newtonian fluids, were obtained experimentally, and found to be in excellent agreement with theoretical predictions. Additionally, a practical application to a DNA solution with incremental ethidium bromide content was employed and the results are consistent with clinical data, indicating that our system provides a potentially important complementary tool for use in such biological and medical applications.
DOI
Prediction of radiation pressure force exerted on moving particles by the two-level skeletonization
Xiao-Min Pan, Ming-Jiang Gou, and Xin-Qing Sheng
A fast full-wave method for computing radiation pressure force (RPF) exerted by shaped light beams on moving particles is presented. The problem of evaluating RPF exerted on a moving particle by a single excitation beam is converted into that of computing RPF’s exerted on a static particle by multiple beams. The discretization of different beams leads to distinct right hand sides (RHS’s) for the matrix system. To avoid solving each RHS by the brute-force manner, the algorithm conducts low-rank decomposition on the excitation matrix consisting of all RHS’s to figure out the so-called skeleton light beams by interpolative decomposition (ID). The peak memory requirement of the skeletonization is a bottle-neck if the particle is large. A two-level skeletonization scheme is proposed to solve this problem. Some numerical experiments on arbitrarily shaped homogeneous particles are performed to illustrate the performance and capability of the developed method.
DOI
A fast full-wave method for computing radiation pressure force (RPF) exerted by shaped light beams on moving particles is presented. The problem of evaluating RPF exerted on a moving particle by a single excitation beam is converted into that of computing RPF’s exerted on a static particle by multiple beams. The discretization of different beams leads to distinct right hand sides (RHS’s) for the matrix system. To avoid solving each RHS by the brute-force manner, the algorithm conducts low-rank decomposition on the excitation matrix consisting of all RHS’s to figure out the so-called skeleton light beams by interpolative decomposition (ID). The peak memory requirement of the skeletonization is a bottle-neck if the particle is large. A two-level skeletonization scheme is proposed to solve this problem. Some numerical experiments on arbitrarily shaped homogeneous particles are performed to illustrate the performance and capability of the developed method.
DOI
Enhancing Nanoparticle Electrodynamics with Gold Nanoplate Mirrors
Zijie Yan , Ying Bao , Uttam Manna , Raman A. Shah , and Norbert F. Scherer
Mirrors and optical cavities can modify and enhance matter–radiation interactions. Here we report that chemically synthesized Au nanoplates can serve as micrometer-size mirrors that enhance electrodynamic interactions. Because of their plasmonic properties, the Au nanoplates enhance the brightness of scattered light from Ag nanoparticles near the nanoplate surface in dark-field microscopy. More importantly, enhanced optical trapping and optical binding of Ag nanoparticles are demonstrated in interferometric optical traps created from a single laser beam and its reflection from individual Au nanoplates. The enhancement of the interparticle force constant is ≈20-fold more than expected from the increased intensity due to standing wave interference. We show that the additional stability for optical binding arises from the restricted axial thermal motion of the nanoparticles that couples to and reduces the fluctuations in the lateral plane. This new mechanism greatly advances the photonic synthesis of ultrastable nanoparticle arrays and investigation of their properties.
DOI
Mirrors and optical cavities can modify and enhance matter–radiation interactions. Here we report that chemically synthesized Au nanoplates can serve as micrometer-size mirrors that enhance electrodynamic interactions. Because of their plasmonic properties, the Au nanoplates enhance the brightness of scattered light from Ag nanoparticles near the nanoplate surface in dark-field microscopy. More importantly, enhanced optical trapping and optical binding of Ag nanoparticles are demonstrated in interferometric optical traps created from a single laser beam and its reflection from individual Au nanoplates. The enhancement of the interparticle force constant is ≈20-fold more than expected from the increased intensity due to standing wave interference. We show that the additional stability for optical binding arises from the restricted axial thermal motion of the nanoparticles that couples to and reduces the fluctuations in the lateral plane. This new mechanism greatly advances the photonic synthesis of ultrastable nanoparticle arrays and investigation of their properties.
DOI
Fiber-integrated optical nano-tweezer based on a bowtie-aperture nano-antenna at the apex of a SNOM tip
Ali El Eter, Nyha M. Hameed, Fadi I. Baida, Roland Salut, Claudine Filiatre, Dusan Nedeljkovic, Elie Atie, Samuel Bole, and Thierry Grosjean
We propose a new concept of fiber-integrated optical nano-tweezer on the basis of a single bowtie-aperture nano-antenna (BNA) fabricated at the apex of a metal-coated SNOM tip. We demonstrate 3D optical trapping of 0.5 micrometer latex beads with input power which does not exceed 1 mW. Optical forces induced by the BNA on tip are then analyzed numerically. They are found to be 103 times larger than the optical forces of a circular aperture of the same area. Such a fiber nanostructure provides a new path for manipulating nano-objects in a compact, flexible and versatile architecture and should thus open promising perspectives in physical, chemical and biomedical domains.
DOI
We propose a new concept of fiber-integrated optical nano-tweezer on the basis of a single bowtie-aperture nano-antenna (BNA) fabricated at the apex of a metal-coated SNOM tip. We demonstrate 3D optical trapping of 0.5 micrometer latex beads with input power which does not exceed 1 mW. Optical forces induced by the BNA on tip are then analyzed numerically. They are found to be 103 times larger than the optical forces of a circular aperture of the same area. Such a fiber nanostructure provides a new path for manipulating nano-objects in a compact, flexible and versatile architecture and should thus open promising perspectives in physical, chemical and biomedical domains.
DOI
A tunable line optical tweezers instrument with nanometer spatial resolution
W. Benjamin Rogers and John C. Crocker
We describe a simple scanning-line optical tweezers instrument for measuring pair interactions between micrometer-sized colloidal particles. Our instrument combines a resonant scanning mirror and an acousto-optic modulator. The resonant scanning mirror creates a time-averaged line trap whose effective one-dimensional intensity profile, and corresponding trapping potential energy landscape can be programmed using the acousto-optic modulator. We demonstrate control over the confining potential by designing and measuring a family of one-dimensional harmonic traps. By adjusting the spring constant, we balance scattering-induced repulsive forces between a pair of trapped particles, creating a flat potential near contact that facilitates interaction measurements. We also develop a simple method for extracting the out-of-plane motion of trapped particles from their relative brightness, allowing us to resolve their relative separation to roughly 1 nm.
DOI
We describe a simple scanning-line optical tweezers instrument for measuring pair interactions between micrometer-sized colloidal particles. Our instrument combines a resonant scanning mirror and an acousto-optic modulator. The resonant scanning mirror creates a time-averaged line trap whose effective one-dimensional intensity profile, and corresponding trapping potential energy landscape can be programmed using the acousto-optic modulator. We demonstrate control over the confining potential by designing and measuring a family of one-dimensional harmonic traps. By adjusting the spring constant, we balance scattering-induced repulsive forces between a pair of trapped particles, creating a flat potential near contact that facilitates interaction measurements. We also develop a simple method for extracting the out-of-plane motion of trapped particles from their relative brightness, allowing us to resolve their relative separation to roughly 1 nm.
DOI
Dynamics of submicron aerosol droplets in a robust optical trap formed by multiple Bessel beams
Ioannis Thanopulos, David Luckhaus, Thomas C. Preston and Ruth Signorell
In this paper, we model the three-dimensional escape dynamics of single submicron-sized aerosol droplets in optical multiple Bessel beam traps. Trapping in counter-propagating Bessel beams (CPBBs) is compared with a newly proposed quadruple Bessel beam (QBB) trap, which consists of two perpendicularly arranged CPBB traps. Calculations are performed for perfectly and imperfectly aligned traps. Mie-theory and finite-difference time-domain methods are used to calculate the optical forces. The droplet escape kinetics are obtained from the solution of the Langevin equation using a Verlet algorithm. Provided the traps are perfectly aligned, the calculations indicate very long lifetimes for droplets trapped either in the CPBB or in the QBB trap. However, minor misalignments that are hard to control experimentally already severely diminish the stability of the CPBB trap. By contrast, such minor misalignments hardly affect the extended droplet lifetimes in a QBB trap. The QBB trap is found to be a stable, robust optical trap, which should enable the experimental investigation of submicron droplets with radii down to 100 nm. Optical binding between two droplets and its potential role in preventing coagulation when loading a CPBB trap is briefly addressed.
DOI
In this paper, we model the three-dimensional escape dynamics of single submicron-sized aerosol droplets in optical multiple Bessel beam traps. Trapping in counter-propagating Bessel beams (CPBBs) is compared with a newly proposed quadruple Bessel beam (QBB) trap, which consists of two perpendicularly arranged CPBB traps. Calculations are performed for perfectly and imperfectly aligned traps. Mie-theory and finite-difference time-domain methods are used to calculate the optical forces. The droplet escape kinetics are obtained from the solution of the Langevin equation using a Verlet algorithm. Provided the traps are perfectly aligned, the calculations indicate very long lifetimes for droplets trapped either in the CPBB or in the QBB trap. However, minor misalignments that are hard to control experimentally already severely diminish the stability of the CPBB trap. By contrast, such minor misalignments hardly affect the extended droplet lifetimes in a QBB trap. The QBB trap is found to be a stable, robust optical trap, which should enable the experimental investigation of submicron droplets with radii down to 100 nm. Optical binding between two droplets and its potential role in preventing coagulation when loading a CPBB trap is briefly addressed.
DOI
Monday, May 5, 2014
Patterned Optical Trapping with Two-Dimensional Photonic Crystals
Peifeng Jing , Jingda Wu , and Lih Y. Lin
We demonstrate an approach to achieve patterned optical trapping with two-dimensional photonic crystals (2D PC). A Gaussian beam infrared laser is guided and loosely focused by a simple optical system onto the surface of the 2D PC, which generates an enhanced diffraction field for optical trapping of microbeads with high efficiency. The diffraction patterns are determined by the structures of the 2D PC, and the experimental results match well with the modeling results using the finite-difference time-domain method. Polarization control is demonstrated as one way to achieve reconfigurability. Using this approach, we demonstrate high design flexibility for patterned optical trapping as determined by the 2D PC structure with low laser intensity.
DOI
We demonstrate an approach to achieve patterned optical trapping with two-dimensional photonic crystals (2D PC). A Gaussian beam infrared laser is guided and loosely focused by a simple optical system onto the surface of the 2D PC, which generates an enhanced diffraction field for optical trapping of microbeads with high efficiency. The diffraction patterns are determined by the structures of the 2D PC, and the experimental results match well with the modeling results using the finite-difference time-domain method. Polarization control is demonstrated as one way to achieve reconfigurability. Using this approach, we demonstrate high design flexibility for patterned optical trapping as determined by the 2D PC structure with low laser intensity.
DOI
Transcription factors TFIIF and TFIIS promote transcript elongation by RNA polymerase II by synergistic and independent mechanisms
Volker Schweikhard, Cong Meng, Kenji Murakami, Craig D. Kaplan, Roger D. Kornberg, and Steven M. Block
Recent evidence suggests that transcript elongation by RNA polymerase II (RNAPII) is regulated by mechanical cues affecting the entry into, and exit from, transcriptionally inactive states, including pausing and arrest. We present a single-molecule optical-trapping study of the interactions of RNAPII with transcription elongation factors TFIIS and TFIIF, which affect these processes. By monitoring the response of elongation complexes containing RNAPII and combinations of TFIIF and TFIIS to controlled mechanical loads, we find that both transcription factors are independently capable of restoring arrested RNAPII to productive elongation. TFIIS, in addition to its established role in promoting transcript cleavage, is found to relieve arrest by a second, cleavage-independent mechanism. TFIIF synergistically enhances some, but not all, of the activities of TFIIS. These studies also uncovered unexpected insights into the mechanisms underlying transient pauses. The direct visualization of pauses at near-base-pair resolution, together with the load dependence of the pause-entry phase, suggests that two distinct mechanisms may be at play: backtracking under forces that hinder transcription and a backtrack-independent activity under assisting loads. The measured pause lifetime distributions are inconsistent with prevailing views of backtracking as a purely diffusive process, suggesting instead that the extent of backtracking may be modulated by mechanisms intrinsic to RNAPII. Pauses triggered by inosine triphosphate misincorporation led to backtracking, even under assisting loads, and their lifetimes were reduced by TFIIS, particularly when aided by TFIIF. Overall, these experiments provide additional insights into how obstacles to transcription may be overcome by the concerted actions of multiple accessory factors.
DOI
Recent evidence suggests that transcript elongation by RNA polymerase II (RNAPII) is regulated by mechanical cues affecting the entry into, and exit from, transcriptionally inactive states, including pausing and arrest. We present a single-molecule optical-trapping study of the interactions of RNAPII with transcription elongation factors TFIIS and TFIIF, which affect these processes. By monitoring the response of elongation complexes containing RNAPII and combinations of TFIIF and TFIIS to controlled mechanical loads, we find that both transcription factors are independently capable of restoring arrested RNAPII to productive elongation. TFIIS, in addition to its established role in promoting transcript cleavage, is found to relieve arrest by a second, cleavage-independent mechanism. TFIIF synergistically enhances some, but not all, of the activities of TFIIS. These studies also uncovered unexpected insights into the mechanisms underlying transient pauses. The direct visualization of pauses at near-base-pair resolution, together with the load dependence of the pause-entry phase, suggests that two distinct mechanisms may be at play: backtracking under forces that hinder transcription and a backtrack-independent activity under assisting loads. The measured pause lifetime distributions are inconsistent with prevailing views of backtracking as a purely diffusive process, suggesting instead that the extent of backtracking may be modulated by mechanisms intrinsic to RNAPII. Pauses triggered by inosine triphosphate misincorporation led to backtracking, even under assisting loads, and their lifetimes were reduced by TFIIS, particularly when aided by TFIIF. Overall, these experiments provide additional insights into how obstacles to transcription may be overcome by the concerted actions of multiple accessory factors.
DOI
Single-molecule studies of riboswitch folding
Andrew Savinov, Christian F. Perez, Steven M. Block
The folding dynamics of riboswitches are central to their ability to modulate gene expression in response to environmental cues. In most cases, a structural competition between the formation of a ligand-binding aptamer and an expression platform (or some other competing off-state) determines the regulatory outcome. Here, we review single-molecule studies of riboswitch folding and function, predominantly carried out using single-molecule FRET or optical trapping approaches. Recent results have supplied new insights into riboswitch folding energy landscapes, the mechanisms of ligand binding, the roles played by divalent ions, the applicability of hierarchical folding models, and kinetic vs. thermodynamic control schemes. We anticipate that future work, based on improved data sets and potentially combining multiple experimental techniques, will enable the development of more complete models for complex RNA folding processes. This article is part of a Special Issue entitled: Riboswitches.
DOI
The folding dynamics of riboswitches are central to their ability to modulate gene expression in response to environmental cues. In most cases, a structural competition between the formation of a ligand-binding aptamer and an expression platform (or some other competing off-state) determines the regulatory outcome. Here, we review single-molecule studies of riboswitch folding and function, predominantly carried out using single-molecule FRET or optical trapping approaches. Recent results have supplied new insights into riboswitch folding energy landscapes, the mechanisms of ligand binding, the roles played by divalent ions, the applicability of hierarchical folding models, and kinetic vs. thermodynamic control schemes. We anticipate that future work, based on improved data sets and potentially combining multiple experimental techniques, will enable the development of more complete models for complex RNA folding processes. This article is part of a Special Issue entitled: Riboswitches.
DOI
Optofluidic rotation of living cells for single-cell tomography
Thorsten Kolb, Sahradha Albert, Michael Haug and Graeme Whyte
Flow cytometry provides a high throughput, multi-dimensional analysis of cells flowing in suspension. In order to combine this feature with the ability to resolve detailed structures in 3D, we developed an optofluidic device that combines a microfluidic system with a dual beam trap. This allows for the rotation of single cells in a continuous flow, around an axis perpendicular to the imaging plane. The combination of both techniques enables the tomographic reconstruction of the 3D structure of the cell. In addition this method is capable to provide detailed 3D structural data for flow cytometry, as it improves the reconstructed z-resolution of a standard microscopy system to produce images with isotropic resolution in all three axes.
DOI
Flow cytometry provides a high throughput, multi-dimensional analysis of cells flowing in suspension. In order to combine this feature with the ability to resolve detailed structures in 3D, we developed an optofluidic device that combines a microfluidic system with a dual beam trap. This allows for the rotation of single cells in a continuous flow, around an axis perpendicular to the imaging plane. The combination of both techniques enables the tomographic reconstruction of the 3D structure of the cell. In addition this method is capable to provide detailed 3D structural data for flow cytometry, as it improves the reconstructed z-resolution of a standard microscopy system to produce images with isotropic resolution in all three axes.
DOI
Sunday, May 4, 2014
Computation of stress on the surface of a soft homogeneous arbitrarily shaped particle
Minglin Yang, Kuan Fang Ren, Yueqian Wu, and Xinqing Sheng
Prediction of the stress on the surface of an arbitrarily shaped particle of soft material is essential in the study of elastic properties of the particles with optical force. It is also necessary in the manipulation and sorting of small particles with optical tweezers, since a regular-shaped particle, such as a sphere, may be deformed under the nonuniform optical stress on its surface. The stress profile on a spherical or small spheroidal soft particle trapped by shaped beams has been studied, however little work on computing the surface stress of an irregular-shaped particle has been reported. We apply in this paper the surface integral equation with multilevel fast multipole algorithm to compute the surface stress on soft homogeneous arbitrarily shaped particles. The comparison of the computed stress profile with that predicted by the generalized Lorenz-Mie theory for a water droplet of diameter equal to 51 wavelengths in a focused Gaussian beam show that the precision of our method is very good. Then stress profiles on spheroids with different aspect ratios are computed. The particles are illuminated by a Gaussian beam of different waist radius at different incidences. Physical analysis on the mechanism of optical stress is given with help of our recently developed vectorial complex ray model. It is found that the maximum of the stress profile on the surface of prolate spheroids is not only determined by the reflected and refracted rays (orders p=0,1) but also the rays undergoing one or two internal reflections where they focus. Computational study of stress on surface of a biconcave cell-like particle, which is a typical application in life science, is also undertaken.
DOI
Prediction of the stress on the surface of an arbitrarily shaped particle of soft material is essential in the study of elastic properties of the particles with optical force. It is also necessary in the manipulation and sorting of small particles with optical tweezers, since a regular-shaped particle, such as a sphere, may be deformed under the nonuniform optical stress on its surface. The stress profile on a spherical or small spheroidal soft particle trapped by shaped beams has been studied, however little work on computing the surface stress of an irregular-shaped particle has been reported. We apply in this paper the surface integral equation with multilevel fast multipole algorithm to compute the surface stress on soft homogeneous arbitrarily shaped particles. The comparison of the computed stress profile with that predicted by the generalized Lorenz-Mie theory for a water droplet of diameter equal to 51 wavelengths in a focused Gaussian beam show that the precision of our method is very good. Then stress profiles on spheroids with different aspect ratios are computed. The particles are illuminated by a Gaussian beam of different waist radius at different incidences. Physical analysis on the mechanism of optical stress is given with help of our recently developed vectorial complex ray model. It is found that the maximum of the stress profile on the surface of prolate spheroids is not only determined by the reflected and refracted rays (orders p=0,1) but also the rays undergoing one or two internal reflections where they focus. Computational study of stress on surface of a biconcave cell-like particle, which is a typical application in life science, is also undertaken.
DOI
Laser wavelength influence on capture and delivery of polystyrene microspheres using nanofibers
Li Ying, Hu Yan-Jun
In the paper we study the effects of laser wavelength on capture and delivery of polystyrene microspheres using a nanofiber. Theoretical analysis shows that when the fiber diameter and the power of the laser are fixed, with the increase of the laser wavelength, the range and intensity of evanescent wave outside optical fiber become large and light gradient and scattering forces exerted on spheres also increase, which means that the ability to capture and transportation of spheres using nanofiber increases with the laser wavelength. The experimental phenomena and theoretical predictions are completely consistent with each other, when the lasers with three wavelengths are separately injected into the fiber with a diameter of 600 nm, with the increase of the wavelength, the critical power becomes small, and when the laser power is constant, with the increase of the wavelength, velocities of spheres also increase, showing that the ability to capture and transport the microsheres is enhanced.
DOI
In the paper we study the effects of laser wavelength on capture and delivery of polystyrene microspheres using a nanofiber. Theoretical analysis shows that when the fiber diameter and the power of the laser are fixed, with the increase of the laser wavelength, the range and intensity of evanescent wave outside optical fiber become large and light gradient and scattering forces exerted on spheres also increase, which means that the ability to capture and transportation of spheres using nanofiber increases with the laser wavelength. The experimental phenomena and theoretical predictions are completely consistent with each other, when the lasers with three wavelengths are separately injected into the fiber with a diameter of 600 nm, with the increase of the wavelength, the critical power becomes small, and when the laser power is constant, with the increase of the wavelength, velocities of spheres also increase, showing that the ability to capture and transport the microsheres is enhanced.
DOI
Trapping of multiple particles by space speckle field and infrared microscopy
Zhi-Gang ZHANG, Feng-Rui LIU, Qing-Chuan ZHANG, Teng CHENG, and Xiao-Ping WU
Optical tweezers technology is widely used in trapping and manipulating micro-and nano-sized particles, mainly including trapping of transparency particles in water and trapping of absorbing particles in air. In this paper, a frosted glass diffuser is irradiated by laser beam, and a subjective speckle field is generated in the image plane of a lens after the laser transmitting the lens. The speckle field is spatial distributed, and contains multiple bright spots and dark spots. A large number of dark spots surrounded by bright spots are spatial energy traps, and can be used to trap a large quantity of absorbing particles. The size and density of trapped particles are about 2~8μm and 1~2g/cm3. In addition, an infrared microscope is used to record the infrared images of the particles trapped by the speckle field, and the infrared images show that the temperature of trapped particles rise by absorbing the light energy, which verifies that the mechanism of trapping absorbing particles by speckle field is photophoretic force.
DOI
Optical tweezers technology is widely used in trapping and manipulating micro-and nano-sized particles, mainly including trapping of transparency particles in water and trapping of absorbing particles in air. In this paper, a frosted glass diffuser is irradiated by laser beam, and a subjective speckle field is generated in the image plane of a lens after the laser transmitting the lens. The speckle field is spatial distributed, and contains multiple bright spots and dark spots. A large number of dark spots surrounded by bright spots are spatial energy traps, and can be used to trap a large quantity of absorbing particles. The size and density of trapped particles are about 2~8μm and 1~2g/cm3. In addition, an infrared microscope is used to record the infrared images of the particles trapped by the speckle field, and the infrared images show that the temperature of trapped particles rise by absorbing the light energy, which verifies that the mechanism of trapping absorbing particles by speckle field is photophoretic force.
DOI
Resonant Pulling of a Microparticle Using a Backward Surface Wave
A. V. Maslov
It is predicted that the optical force experienced by a dielectric particle excited resonantly by a surface wave can be directed opposite to the incident power flow when the exciting wave is a backward one. This is consistent with the electromagnetic momentum flow of the backward wave being directed opposite to the power flow. The magnitude of the force can be comparable to the momentum flow of the surface wave. Such forces bring a deeper understanding of the electromagnetics of backward surface waves and can be used in integrated photonic circuits and optofluidic devices.
DOI
It is predicted that the optical force experienced by a dielectric particle excited resonantly by a surface wave can be directed opposite to the incident power flow when the exciting wave is a backward one. This is consistent with the electromagnetic momentum flow of the backward wave being directed opposite to the power flow. The magnitude of the force can be comparable to the momentum flow of the surface wave. Such forces bring a deeper understanding of the electromagnetics of backward surface waves and can be used in integrated photonic circuits and optofluidic devices.
DOI
Inhomogeneous and anisotropic particles in optical traps: Physical behaviour and applications
S.H. Simpson
Beyond the ubiquitous colloidal sphere, optical tweezers are capable of trapping myriad exotic particles with wildly varying geometries and compositions. This simple fact opens up numerous opportunities for micro-manipulation, directed assembly and characterization of novel nanostructures. Furthermore, the mechanical properties of optical tweezers are transformed by their contents. For example, traps capable of measuring, or applying, femto-Newton scale forces with nanometric spatial resolution can be designed. Analogous, if not superior, angular sensitivity can be achieved, enabling the creation of exquisitely sensitive torque wrenches. These capacities, and others, lead to a multitude of novel applications in the meso- and nanosciences. In this article we review experimental and theoretical work on the relationship between particle geometry, composition and trap properties. A range of associated metrological techniques are discussed.
DOI
Beyond the ubiquitous colloidal sphere, optical tweezers are capable of trapping myriad exotic particles with wildly varying geometries and compositions. This simple fact opens up numerous opportunities for micro-manipulation, directed assembly and characterization of novel nanostructures. Furthermore, the mechanical properties of optical tweezers are transformed by their contents. For example, traps capable of measuring, or applying, femto-Newton scale forces with nanometric spatial resolution can be designed. Analogous, if not superior, angular sensitivity can be achieved, enabling the creation of exquisitely sensitive torque wrenches. These capacities, and others, lead to a multitude of novel applications in the meso- and nanosciences. In this article we review experimental and theoretical work on the relationship between particle geometry, composition and trap properties. A range of associated metrological techniques are discussed.
DOI
Programmed ribosomal frameshifting occurs at multiple sites and by multiple paths
Ignacio Tinoco Jr., Shannon Yan and Hee-Kyung Kim
Programmed frameshifting is used by prokaryotes and eukaryotes to synthesize two or more proteins from the same messenger RNA. We have studied minus-one frameshifting in the dnaX gene in E. coli, whose mRNA contains the usual frameshifting signals: an internal Shine Dalgarno sequence, a slippery sequence (AAAAAAG), and a stem-loop. We used bulk mass spectrometry, single-molecule laser tweezers, and single-molecule FRET in our studies. We found that minus-one frameshifting occurs at the Lys codons in the slippery sequence, but also at codons on either side. Furthermore, the minus-one frameshift occurs by slips of the ribosome of -1, +2, or even -4 nucleotides. The translation trajectories show step-by-step progression as each codon is translated, but at the slippery sequence large-scale fluctuations in position of the ribosome are seen. This agrees with the mass spectrometry results showing multiple sites and multiple paths of frameshifting. After peptide bond formation the tRNAs undergo a classic-hybrid equilibrium before the elongation factor, EF-G•GTP, catalyzes the translocation. Our single-molecule FRET studies of fluorophore-labeled ribosome and tRNA found that the presence of the stem-loop stabilizes the ribosome in the hybrid state before translocation. The longer time spent in this state allows the ribosome to sample other states, and thus may favor sites and paths in addition to a zero-frame move.
DOI
Programmed frameshifting is used by prokaryotes and eukaryotes to synthesize two or more proteins from the same messenger RNA. We have studied minus-one frameshifting in the dnaX gene in E. coli, whose mRNA contains the usual frameshifting signals: an internal Shine Dalgarno sequence, a slippery sequence (AAAAAAG), and a stem-loop. We used bulk mass spectrometry, single-molecule laser tweezers, and single-molecule FRET in our studies. We found that minus-one frameshifting occurs at the Lys codons in the slippery sequence, but also at codons on either side. Furthermore, the minus-one frameshift occurs by slips of the ribosome of -1, +2, or even -4 nucleotides. The translation trajectories show step-by-step progression as each codon is translated, but at the slippery sequence large-scale fluctuations in position of the ribosome are seen. This agrees with the mass spectrometry results showing multiple sites and multiple paths of frameshifting. After peptide bond formation the tRNAs undergo a classic-hybrid equilibrium before the elongation factor, EF-G•GTP, catalyzes the translocation. Our single-molecule FRET studies of fluorophore-labeled ribosome and tRNA found that the presence of the stem-loop stabilizes the ribosome in the hybrid state before translocation. The longer time spent in this state allows the ribosome to sample other states, and thus may favor sites and paths in addition to a zero-frame move.
DOI
Friday, May 2, 2014
Asymmetric Friction of Nonmotor MAPs Can Lead to Their Directional Motion in Active Microtubule Networks
Scott Forth, Kuo-Chiang Hsia, Yuta Shimamoto, Tarun M. Kapoor
Diverse cellular processes require microtubules to be organized into distinct structures, such as asters or bundles. Within these dynamic motifs, microtubule-associated proteins (MAPs) are frequently under load, but how force modulates these proteins’ function is poorly understood. Here, we combine optical trapping with TIRF-based microscopy to measure the force dependence of microtubule interaction for three nonmotor MAPs (NuMA, PRC1, and EB1) required for cell division. We find that frictional forces increase nonlinearly with MAP velocity across microtubules and depend on filament polarity, with NuMA’s friction being lower when moving toward minus ends, EB1’s lower toward plus ends, and PRC1's exhibiting no directional preference. Mathematical models predict, and experiments confirm, that MAPs with asymmetric friction can move directionally within actively moving microtubule pairs they crosslink. Our findings reveal how nonmotor MAPs can generate frictional resistance in dynamic cytoskeletal networks via micromechanical adaptations whose anisotropy may be optimized for MAP localization and function within cellular structures.
DOI
Diverse cellular processes require microtubules to be organized into distinct structures, such as asters or bundles. Within these dynamic motifs, microtubule-associated proteins (MAPs) are frequently under load, but how force modulates these proteins’ function is poorly understood. Here, we combine optical trapping with TIRF-based microscopy to measure the force dependence of microtubule interaction for three nonmotor MAPs (NuMA, PRC1, and EB1) required for cell division. We find that frictional forces increase nonlinearly with MAP velocity across microtubules and depend on filament polarity, with NuMA’s friction being lower when moving toward minus ends, EB1’s lower toward plus ends, and PRC1's exhibiting no directional preference. Mathematical models predict, and experiments confirm, that MAPs with asymmetric friction can move directionally within actively moving microtubule pairs they crosslink. Our findings reveal how nonmotor MAPs can generate frictional resistance in dynamic cytoskeletal networks via micromechanical adaptations whose anisotropy may be optimized for MAP localization and function within cellular structures.
DOI
Highly Effective Photonic Cue for Repulsive Axonal Guidance
Bryan J. Black, Ling Gu, Samarendra K. Mohanty
In vivo nerve repair requires not only the ability to regenerate damaged axons, but most importantly, the ability to guide developing or regenerating axons along paths that will result in functional connections. Furthermore, basic studies in neuroscience and neuro-electronic interface design require the ability to construct in vitro neural circuitry. Both these applications require the development of a noninvasive, highly effective tool for axonal growth-cone guidance. To date, a myriad of technologies have been introduced based on chemical, electrical, mechanical, and hybrid approaches (such as electro-chemical, optofluidic flow and photo-chemical methods). These methods are either lacking in desired spatial and temporal selectivity or require the introduction of invasive external factors. Within the last fifteen years however, several attractive guidance cues have been developed using purely light based cues to achieve axonal guidance. Here, we report a novel, purely optical repulsive guidance technique that uses low power, near infrared light, and demonstrates the guidance of primary goldfish retinal ganglion cell axons through turns of up to 120 degrees and over distances of ~90 µm.
DOI
In vivo nerve repair requires not only the ability to regenerate damaged axons, but most importantly, the ability to guide developing or regenerating axons along paths that will result in functional connections. Furthermore, basic studies in neuroscience and neuro-electronic interface design require the ability to construct in vitro neural circuitry. Both these applications require the development of a noninvasive, highly effective tool for axonal growth-cone guidance. To date, a myriad of technologies have been introduced based on chemical, electrical, mechanical, and hybrid approaches (such as electro-chemical, optofluidic flow and photo-chemical methods). These methods are either lacking in desired spatial and temporal selectivity or require the introduction of invasive external factors. Within the last fifteen years however, several attractive guidance cues have been developed using purely light based cues to achieve axonal guidance. Here, we report a novel, purely optical repulsive guidance technique that uses low power, near infrared light, and demonstrates the guidance of primary goldfish retinal ganglion cell axons through turns of up to 120 degrees and over distances of ~90 µm.
DOI
On the Effects of Intercalators in DNA Condensation: A Force Spectroscopy and Gel Electrophoresis Study
M. S. Rocha , A. G. Cavalcante , R. Silva , and E. B. Ramos
In this work we have characterized the effects of the intercalator ethidium bromide (EtBr) on the DNA condensation process by using force spectroscopy and gel electrophoresis. We have tested two condensing agents: spermine (spm4+), a tetravalent cationic amine which promotes cation-induced DNA condensation, and poly(ethylene glycol) (PEG), a neutral polymer which promotes DNA ψ-condensation. Two different types of experiments were performed. In the first type, bare DNA molecules disperse in solution are first treated with EtBr for intercalation, and then the condensing agent is added to the sample with the purpose of verifying the effects of the intercalator in hindering DNA condensation. In the second experiment type, the bare DNA molecules are first condensed, and then the intercalator is added to the sample in order to verify its influence on the previously condensed DNA. The results obtained with the two different experimental techniques used agree very well, indicating that previously intercalated EtBr can hinder both cation-induced and ψ-condensation, being more efficient in the first case. On the other hand, EtBr has little effect on the previously formed cation-induced condensates, but is efficient in unfolding the ψ-condensates.
DOI
In this work we have characterized the effects of the intercalator ethidium bromide (EtBr) on the DNA condensation process by using force spectroscopy and gel electrophoresis. We have tested two condensing agents: spermine (spm4+), a tetravalent cationic amine which promotes cation-induced DNA condensation, and poly(ethylene glycol) (PEG), a neutral polymer which promotes DNA ψ-condensation. Two different types of experiments were performed. In the first type, bare DNA molecules disperse in solution are first treated with EtBr for intercalation, and then the condensing agent is added to the sample with the purpose of verifying the effects of the intercalator in hindering DNA condensation. In the second experiment type, the bare DNA molecules are first condensed, and then the intercalator is added to the sample in order to verify its influence on the previously condensed DNA. The results obtained with the two different experimental techniques used agree very well, indicating that previously intercalated EtBr can hinder both cation-induced and ψ-condensation, being more efficient in the first case. On the other hand, EtBr has little effect on the previously formed cation-induced condensates, but is efficient in unfolding the ψ-condensates.
DOI
Label-Free Free-Solution Single-Molecule Protein–Small Molecule Interaction Observed by Double-Nanohole Plasmonic Trapping
Ahmed A. Al Balushi and Reuven Gordon
The interaction of proteins with small molecules is fundamental to their function in living organisms, and it is widely studied in drug development. Here we use the double nanohole optical trapping technique to observe real-time label-free free-solution single-molecule dynamics of three complexes: biotin–streptavidin, biotin–monovalent streptavidin, and acetylsalicylic acid–cyclooxygenase 2. Radically different behavior is seen between the protein with and without the small molecule binding. This detection platform is scalable, inexpensive, and highly sensitive, which may transform drug discovery based on protein–small molecule interactions.
DOI
The interaction of proteins with small molecules is fundamental to their function in living organisms, and it is widely studied in drug development. Here we use the double nanohole optical trapping technique to observe real-time label-free free-solution single-molecule dynamics of three complexes: biotin–streptavidin, biotin–monovalent streptavidin, and acetylsalicylic acid–cyclooxygenase 2. Radically different behavior is seen between the protein with and without the small molecule binding. This detection platform is scalable, inexpensive, and highly sensitive, which may transform drug discovery based on protein–small molecule interactions.
DOI
Resonant optical gun
A. V. Maslov and M. I. Bakunov
We propose a concept of a structure—a resonant optical gun—to realize an efficient propulsion of dielectric microparticles by light forces. The structure is based on a waveguide in which a reversal of the electromagnetic momentum flow of the incident mode is realized by exciting a whispering gallery resonance in the microparticle. The propelling force can reach the value up to the theoretical maximum of twice the momentum flow of the initial wave. The force density oscillates along the particle periphery and has very large amplitude.
DOI
We propose a concept of a structure—a resonant optical gun—to realize an efficient propulsion of dielectric microparticles by light forces. The structure is based on a waveguide in which a reversal of the electromagnetic momentum flow of the incident mode is realized by exciting a whispering gallery resonance in the microparticle. The propelling force can reach the value up to the theoretical maximum of twice the momentum flow of the initial wave. The force density oscillates along the particle periphery and has very large amplitude.
DOI
Engineering light-matter interaction for emerging optical manipulation applications
Cheng-Wei Qiu, Darwin Palima, Andrey Novitsky, Dongliang Gao, Weiqiang Ding, Sergei V. Zhukovsky, Jesper Gluckstad
In this review, we explore recent trends in optical micromanipulation by engineering light-matter interaction and controlling the mechanical effects of optical fields. One central theme is exploring the rich phenomena beyond the now established precision measurements based on trapping micro beads with tightly focused beams. Novel synthesized beams, exploiting the linear and angular momentum of light, open new possibilities in optical trapping and micromanipulation. Similarly, novel structures are promising to enable new optical micromanipulation modalities. Moreover, an overview of the amazing features of the optics of tractor beams and backward-directed energy fluxes will be presented. Recently the so-called effect of negative propagation of the beams (existence of the backward energy fluxes) has been confirmed for X-waves and Airy beams. In the review, we will also discuss the negative pulling force of structured beams and negative energy fluxes in the vicinity of fibers. The effect is achieved due to the interaction of multipoles or, in another interpretation, the momentum conservation. Both backward-directed Poynting vector and backward optical forces are counter-intuitive and give an insight into new physics and technologies. Exploiting the degrees of freedom in synthesizing novel beams and designed microstructures offer attractive prospects for emerging optical manipulation applications.
DOI
In this review, we explore recent trends in optical micromanipulation by engineering light-matter interaction and controlling the mechanical effects of optical fields. One central theme is exploring the rich phenomena beyond the now established precision measurements based on trapping micro beads with tightly focused beams. Novel synthesized beams, exploiting the linear and angular momentum of light, open new possibilities in optical trapping and micromanipulation. Similarly, novel structures are promising to enable new optical micromanipulation modalities. Moreover, an overview of the amazing features of the optics of tractor beams and backward-directed energy fluxes will be presented. Recently the so-called effect of negative propagation of the beams (existence of the backward energy fluxes) has been confirmed for X-waves and Airy beams. In the review, we will also discuss the negative pulling force of structured beams and negative energy fluxes in the vicinity of fibers. The effect is achieved due to the interaction of multipoles or, in another interpretation, the momentum conservation. Both backward-directed Poynting vector and backward optical forces are counter-intuitive and give an insight into new physics and technologies. Exploiting the degrees of freedom in synthesizing novel beams and designed microstructures offer attractive prospects for emerging optical manipulation applications.
DOI
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