Nir Katchinskiy, Roseline Godbout, Helly R. Goez, Abdulhakem Y. Elezzabi
We have demonstrated the ability to attach single cells using sub-10 femtosecond laser pulses, with 800 nm central wavelength delivered from a Ti:Sapphire laser. To check that the cells did not go through a cell-fusion process, a fluorescent dye Calcein AM was used to verify that the fluorescent dye did not migrate from a dyed cell to a non-dyed cell. The mechanical integrity of the attached joint was assessed using an optical tweezer. Attachment of cells was performed without the induction of cell-cell fusion, with attachment efficiency of 95%, and while preserving the cells' viability. Cell-cell attachment was achieved by delivery of one to two trains of femtosecond laser pulses lasting 15 ms each. Laser-induced ionization process led to an ultrafast reversible destabilization of the phospholipid layer of the cellular membrane. The inner cell membrane remained intact during the attachment procedure, and isolation of the cells' cytoplasm from the surrounding medium was obtained. A strong physical attachment between the cells was obtained due to the bonding of the membranes' ionized phospholipid molecules and the formation of a joint cellular membrane at the connection point. The cellular attachment technique, femtosecond laser-induced cell-cell surgical attachment, can potentially provide a platform for the creation of engineered tissue and cell cultures.
DOI
Monday, February 24, 2014
Plasmonic particles set into fast orbital motion by an optical vortex beam
Anni Lehmuskero, Yanming Li, Peter Johansson, and Mikael Käll
We optically trap plasmonic gold particles in two dimensions and set them into circular motion around the optical axis using a helically phased vortex laser beam. The orbiting frequency of the particles reaches 86 Hz, which corresponds to a particle velocity of the order 1 mm per second, for an incident laser power of a few tens of milliwatts. The experimentally determined orbiting frequencies are found to be well in line with the notion that the beam carries an orbital angular momentum of ħl per photon.
DOI
We optically trap plasmonic gold particles in two dimensions and set them into circular motion around the optical axis using a helically phased vortex laser beam. The orbiting frequency of the particles reaches 86 Hz, which corresponds to a particle velocity of the order 1 mm per second, for an incident laser power of a few tens of milliwatts. The experimentally determined orbiting frequencies are found to be well in line with the notion that the beam carries an orbital angular momentum of ħl per photon.
DOI
Holographic three-dimensional motion detection of an optically trapped sub-100 nm gold nanoparticle
Yoshio Hayasaki, Akira Sato
We demonstrated, for the first time, three-dimensional (3D) motion detection of a gold nanoparticle held in optical tweezers in water using holographic microscopy. Motion detection was performed with an in-line, low-coherence digital holographic microscope. The nanoparticle diameter was 60 nm, which is the smallest reported particle whose 3D motion can be measured. The motion of the optically-trapped nanoparticle had an axial variation of 7.6 nm (standard deviation) when a 1070 nm laser beam with an intensity of more than 27 MW/cm2 was focused with a 1.25 NA objective lens. From a comparison of a gold nanoparticle fixed on a glass substrate and a gold nanoparticle trapped by optical tweezers with a sufficient light intensity, we found that most of the variation was caused by noise in the experimental setup, and not the motion itself. The lateral variations for the fixed and trapped nanoparticles were the same, but the axial variation for the trapped nanoparticle was slightly larger. This suggested that the optically trapped nanoparticle had a motion with a single-nanometer order in the standard deviation only along the axial direction when the laser intensity was sufficiently large for trapping, which in this experiment was more than 27 MW/cm2.
DOI
We demonstrated, for the first time, three-dimensional (3D) motion detection of a gold nanoparticle held in optical tweezers in water using holographic microscopy. Motion detection was performed with an in-line, low-coherence digital holographic microscope. The nanoparticle diameter was 60 nm, which is the smallest reported particle whose 3D motion can be measured. The motion of the optically-trapped nanoparticle had an axial variation of 7.6 nm (standard deviation) when a 1070 nm laser beam with an intensity of more than 27 MW/cm2 was focused with a 1.25 NA objective lens. From a comparison of a gold nanoparticle fixed on a glass substrate and a gold nanoparticle trapped by optical tweezers with a sufficient light intensity, we found that most of the variation was caused by noise in the experimental setup, and not the motion itself. The lateral variations for the fixed and trapped nanoparticles were the same, but the axial variation for the trapped nanoparticle was slightly larger. This suggested that the optically trapped nanoparticle had a motion with a single-nanometer order in the standard deviation only along the axial direction when the laser intensity was sufficiently large for trapping, which in this experiment was more than 27 MW/cm2.
DOI
Mechanical separation of chiral dipoles by chiral light
Antoine Canaguier-Durand, James A Hutchison, Cyriaque Genet1 and Thomas W Ebbesen
We calculate optical forces and torques exerted on a chiral dipole by chiral light fields and reveal genuine chiral forces in combining the chiral contents of both light field and dipolar matter. Here, the optical chirality is characterized in a general way through the definition of optical chirality density and chirality flow. We show, in particular, that both terms have mechanical effects associated, respectively, with reactive and dissipative components of the chiral forces. Remarkably, these chiral force components are directly related to standard observables: optical rotation for the reactive component and circular dichroism for the dissipative one. As a consequence, the resulting forces and torques are dependent on the enantiomeric form of the chiral dipole. This suggests promising strategies for using chiral light forces to mechanically separate chiral objects according to their enantiomeric form.
DOI
We calculate optical forces and torques exerted on a chiral dipole by chiral light fields and reveal genuine chiral forces in combining the chiral contents of both light field and dipolar matter. Here, the optical chirality is characterized in a general way through the definition of optical chirality density and chirality flow. We show, in particular, that both terms have mechanical effects associated, respectively, with reactive and dissipative components of the chiral forces. Remarkably, these chiral force components are directly related to standard observables: optical rotation for the reactive component and circular dichroism for the dissipative one. As a consequence, the resulting forces and torques are dependent on the enantiomeric form of the chiral dipole. This suggests promising strategies for using chiral light forces to mechanically separate chiral objects according to their enantiomeric form.
DOI
Saturday, February 22, 2014
Theoretical analysis for the optical deformation of emulsion droplets
David Tapp, Jonathan M. Taylor, Alex S. Lubansky, Colin D. Bain, and Buddhapriya Chakrabarti
We propose a theoretical framework to predict the three-dimensional shapes of optically deformed micron-sized emulsion droplets with ultra-low interfacial tension. The resulting shape and size of the droplet arises out of a balance between the interfacial tension and optical forces. Using an approximation of the laser field as a Gaussian beam, working within the Rayleigh-Gans regime and assuming isotropic surface energy at the oil-water interface, we numerically solve the resulting shape equations to elucidate the three-dimensional droplet geometry. We obtain a plethora of shapes as a function of the number of optical tweezers, their laser powers and positions, surface tension, initial droplet size and geometry. Experimentally, two-dimensional droplet silhouettes have been imaged from above, but their full side-on view has not been observed and reported for current optical configurations. This experimental limitation points to ambiguity in differentiating between droplets having the same two-dimensional projection but with disparate three-dimensional shapes. Our model elucidates and quantifies this difference for the first time. We also provide a dimensionless number that indicates the shape transformation (ellipsoidal to dumbbell) at a value ≈ 1.0, obtained by balancing interfacial tension and laser forces, substantiated using a data collapse.
DOI
We propose a theoretical framework to predict the three-dimensional shapes of optically deformed micron-sized emulsion droplets with ultra-low interfacial tension. The resulting shape and size of the droplet arises out of a balance between the interfacial tension and optical forces. Using an approximation of the laser field as a Gaussian beam, working within the Rayleigh-Gans regime and assuming isotropic surface energy at the oil-water interface, we numerically solve the resulting shape equations to elucidate the three-dimensional droplet geometry. We obtain a plethora of shapes as a function of the number of optical tweezers, their laser powers and positions, surface tension, initial droplet size and geometry. Experimentally, two-dimensional droplet silhouettes have been imaged from above, but their full side-on view has not been observed and reported for current optical configurations. This experimental limitation points to ambiguity in differentiating between droplets having the same two-dimensional projection but with disparate three-dimensional shapes. Our model elucidates and quantifies this difference for the first time. We also provide a dimensionless number that indicates the shape transformation (ellipsoidal to dumbbell) at a value ≈ 1.0, obtained by balancing interfacial tension and laser forces, substantiated using a data collapse.
DOI
Geometrically unrestricted, topologically constrained control of liquid crystal defects using simultaneous holonomic magnetic and holographic optical manipulation
Michael C. M. Varney, Nathan J. Jenness, and Ivan I. Smalyukh
Despite the recent progress in physical control and manipulation of various condensed matter, atomic, and particle systems, including individual atoms and photons, our ability to control topological defects remains limited. Recently, controlled generation, spatial translation, and stretching of topological point and line defects have been achieved using laser tweezers and liquid crystals as model defect-hosting systems. However, many modes of manipulation remain hindered by limitations inherent to optical trapping. To overcome some of these limitations, we integrate holographic optical tweezers with a magnetic manipulation system, which enables fully holonomic manipulation of defects by means of optically and magnetically controllable colloids used as “handles” to transfer forces and torques to various liquid crystal defects. These colloidal handles are magnetically rotated around determined axes and are optically translated along three-dimensional pathways while mechanically attached to defects, which, combined with inducing spatially localized nematic-isotropic phase transitions, allow for geometrically unrestricted control of defects, including previously unrealized modes of noncontact manipulation, such as the twisting of disclination clusters. These manipulation capabilities may allow for probing topological constraints and the nature of defects in unprecedented ways, providing the foundation for a tabletop laboratory to expand our understanding of the role defects play in fields ranging from subatomic particle physics to early-universe cosmology.
DOI
Despite the recent progress in physical control and manipulation of various condensed matter, atomic, and particle systems, including individual atoms and photons, our ability to control topological defects remains limited. Recently, controlled generation, spatial translation, and stretching of topological point and line defects have been achieved using laser tweezers and liquid crystals as model defect-hosting systems. However, many modes of manipulation remain hindered by limitations inherent to optical trapping. To overcome some of these limitations, we integrate holographic optical tweezers with a magnetic manipulation system, which enables fully holonomic manipulation of defects by means of optically and magnetically controllable colloids used as “handles” to transfer forces and torques to various liquid crystal defects. These colloidal handles are magnetically rotated around determined axes and are optically translated along three-dimensional pathways while mechanically attached to defects, which, combined with inducing spatially localized nematic-isotropic phase transitions, allow for geometrically unrestricted control of defects, including previously unrealized modes of noncontact manipulation, such as the twisting of disclination clusters. These manipulation capabilities may allow for probing topological constraints and the nature of defects in unprecedented ways, providing the foundation for a tabletop laboratory to expand our understanding of the role defects play in fields ranging from subatomic particle physics to early-universe cosmology.
DOI
Dynamic Trapping and Two-Dimensional Transport of Swimming Microorganisms Using a Rotating Magnetic Micro-Robot
Zhou Ye and Metin Sitti
Manipulation of microorganisms with intrinsic motility is a challenging yet important task for many biological and biomedical applications. Currently, such a task has only been accomplished using optical tweezers, while at the risk of averse heating and photodamage of the biological samples. Here we proposed a new micro-robotic approach for fluidic trapping and two-dimensional transportation of motile microorganisms near a solid surface in fluids. We demonstrated selective trapping and transportation of individual freely swimming multi-flagellated bacteria over a distance of 30 μm (10 bodylength of the particle) on a surface, using the rotational flows locally induced by a rotating magnetic micro-particle. Only a weak uniform magnetic field (< 3 mT) was applied to actuate the micro-particle. The micro-particle can translate on a glass substrate by rotation at a speed of up to 100 μm/s, while providing a fluidic force of a few to tens of pico-Newtons.
DOI
Manipulation of microorganisms with intrinsic motility is a challenging yet important task for many biological and biomedical applications. Currently, such a task has only been accomplished using optical tweezers, while at the risk of averse heating and photodamage of the biological samples. Here we proposed a new micro-robotic approach for fluidic trapping and two-dimensional transportation of motile microorganisms near a solid surface in fluids. We demonstrated selective trapping and transportation of individual freely swimming multi-flagellated bacteria over a distance of 30 μm (10 bodylength of the particle) on a surface, using the rotational flows locally induced by a rotating magnetic micro-particle. Only a weak uniform magnetic field (< 3 mT) was applied to actuate the micro-particle. The micro-particle can translate on a glass substrate by rotation at a speed of up to 100 μm/s, while providing a fluidic force of a few to tens of pico-Newtons.
DOI
Optical trap formation with a four-channel liquid crystal light modulator
A V Korobtsov, S P Kotova, N N Losevsky, A M Mayorova, V V Patlan and S A Samagin
Experiments on the dynamic control of two-dimensional optical trap spatial structure and manipulation of micron-sized dielectric particles are performed with a four-channel liquid crystal modulator. Tweezers with this device can execute the trapping of a single particle and its subsequent relocation along the square trajectory. Also, when shaped as a line segment, the trap can capture several particles and rotate them. Therefore, it is experimentally proven that for certain tasks this device can be a compact and technologically simple alternative to the available commercial multi-pixel liquid crystal modulators.
DOI
Experiments on the dynamic control of two-dimensional optical trap spatial structure and manipulation of micron-sized dielectric particles are performed with a four-channel liquid crystal modulator. Tweezers with this device can execute the trapping of a single particle and its subsequent relocation along the square trajectory. Also, when shaped as a line segment, the trap can capture several particles and rotate them. Therefore, it is experimentally proven that for certain tasks this device can be a compact and technologically simple alternative to the available commercial multi-pixel liquid crystal modulators.
DOI
Tuning the size and configuration of nanocarbon microcapsules: aqueous method using optical tweezers
Hiroshi Frusawa & Youei Matsumoto
To date, optical manipulation techniques for aqueous dispersions have been developed that deposit and/or transport nanoparticles not only for fundamental studies of colloidal dynamics, but also for either creating photonic devices or allowing accurate control of liquids on micron scales. Here, we report that optical tweezers (OT) system is able to direct three-dimensional assembly of graphene, graphite, and carbon nanotubes (CNT) into microcapsules of hollow spheres. The OT technique facilitates both to visualize the elasticity of a CNT microcapsule and to arrange a triplet of identical graphene microcapsules in aqueous media. Furthermore, the similarity of swelling courses has been found over a range of experimental parameters such as nanocarbon species, the power of the incident light, and the suspension density. Thanks to the universality in evolutions of rescaled capsule size, we can precisely control the size of various nanocarbon microcapsules by adjusting the duration time of laser emission.
DOI
To date, optical manipulation techniques for aqueous dispersions have been developed that deposit and/or transport nanoparticles not only for fundamental studies of colloidal dynamics, but also for either creating photonic devices or allowing accurate control of liquids on micron scales. Here, we report that optical tweezers (OT) system is able to direct three-dimensional assembly of graphene, graphite, and carbon nanotubes (CNT) into microcapsules of hollow spheres. The OT technique facilitates both to visualize the elasticity of a CNT microcapsule and to arrange a triplet of identical graphene microcapsules in aqueous media. Furthermore, the similarity of swelling courses has been found over a range of experimental parameters such as nanocarbon species, the power of the incident light, and the suspension density. Thanks to the universality in evolutions of rescaled capsule size, we can precisely control the size of various nanocarbon microcapsules by adjusting the duration time of laser emission.
DOI
Thursday, February 20, 2014
Joining forces: integrating the mechanical and optical single molecule toolkits
Monique J. Jacobs and Kerstin Blank
Single molecule force spectroscopy and single molecule fluorescence microscopy have both evolved into extremely powerful tools for studying molecular mechanisms in Biophysics and Materials Science. Recent technological developments have focused on combining the strengths of both techniques in one instrument. Integrated instruments provide unique possibilities for mechanically manipulating a single molecule while observing its response optically. Here we provide an overview of the state-of-the-art with an emphasis on the technological challenges. Describing the mostly biological systems that have been studied to date, we discuss the application of combined force-fluorescence approaches for studying force–structure–function relationships. We further highlight the potential of integrated setups for investigating mechanosensing and mechanoregulation in biological systems and for characterizing molecular force probes that find potential application in (biomimetic) self-reporting and self-healing materials.
DOI
Single molecule force spectroscopy and single molecule fluorescence microscopy have both evolved into extremely powerful tools for studying molecular mechanisms in Biophysics and Materials Science. Recent technological developments have focused on combining the strengths of both techniques in one instrument. Integrated instruments provide unique possibilities for mechanically manipulating a single molecule while observing its response optically. Here we provide an overview of the state-of-the-art with an emphasis on the technological challenges. Describing the mostly biological systems that have been studied to date, we discuss the application of combined force-fluorescence approaches for studying force–structure–function relationships. We further highlight the potential of integrated setups for investigating mechanosensing and mechanoregulation in biological systems and for characterizing molecular force probes that find potential application in (biomimetic) self-reporting and self-healing materials.
DOI
Wednesday, February 19, 2014
Non-destructive handling of individual chromatin fibers isolated from single cells in a microfluidic device utilizing an optically driven microtool
Hidehiro Oana, Kaori Nishikawa, Hirotada Matsuhara, Ayumu Yamamoto, Takaharu G. Yamamoto, Tokuko Haraguchi, Yasushi Hiraoka and Masao Washizu
We report a novel method for the non-destructive handling of, and biochemical experiments with, individual intact chromatin fibers, as well as their isolation from single cells, utilizing a specifically designed microfluidic device with an optically driven microtool under the microscope. Spheroplasts of recombinant fission yeast cells expressing fluorescent protein-tagged core histones were employed, and isolation of chromatin fibers was conducted by cell bursting via changing from isotonic conditions to hypotonic conditions in the microfluidic device. The isolation of chromatin fibers was confirmed by the fluorescent protein-tagged core histones involved in the chromatin fibers. For the non-destructive handling of the isolated chromatin fibers in the microfluidic device, we developed antibody-conjugated microspheres, which had affinity to the fluorescent protein-tagged core histones, and the microspheres were manipulated using optical tweezers, which functioned as optically driven microtools. With the aid of the microtool, isolated chromatin fibers were handled non-destructively and were tethered at the microstructures fabricated in the microfluidic device with straightened conformation by the flow. Immunofluorescence staining was carried out as a demonstrative biochemical experiment with the individual native chromatin fibers isolated in the microfluidic device, and specific fluorescent spots were visualized along the tethered chromatin fibers. Thus, the potential application of this method for epigenetic analyses of intact chromatin fibers isolated from single cells is demonstrated.
DOI
We report a novel method for the non-destructive handling of, and biochemical experiments with, individual intact chromatin fibers, as well as their isolation from single cells, utilizing a specifically designed microfluidic device with an optically driven microtool under the microscope. Spheroplasts of recombinant fission yeast cells expressing fluorescent protein-tagged core histones were employed, and isolation of chromatin fibers was conducted by cell bursting via changing from isotonic conditions to hypotonic conditions in the microfluidic device. The isolation of chromatin fibers was confirmed by the fluorescent protein-tagged core histones involved in the chromatin fibers. For the non-destructive handling of the isolated chromatin fibers in the microfluidic device, we developed antibody-conjugated microspheres, which had affinity to the fluorescent protein-tagged core histones, and the microspheres were manipulated using optical tweezers, which functioned as optically driven microtools. With the aid of the microtool, isolated chromatin fibers were handled non-destructively and were tethered at the microstructures fabricated in the microfluidic device with straightened conformation by the flow. Immunofluorescence staining was carried out as a demonstrative biochemical experiment with the individual native chromatin fibers isolated in the microfluidic device, and specific fluorescent spots were visualized along the tethered chromatin fibers. Thus, the potential application of this method for epigenetic analyses of intact chromatin fibers isolated from single cells is demonstrated.
DOI
A new approach to determine vapour pressures and hygroscopicities of aqueous aerosols containing semi-volatile organic compounds
C. Cai, D. J. Stewart, T. C. Preston, J. S. Walker, Y.-H. Zhang and J. P. Reid
We present a new approach to study the equilibrium gas-particle partitioning of volatile and semi-volatile organic components in aqueous aerosol, deriving a correlational analysis method that examines and interprets simultaneous and correlated fluctuations in particle size and composition. From this approach, changes in particle size driven by organic component evaporation can be clearly resolved from size changes driven by hygroscopicity and fluctuations in environmental conditions. The approach is used to interpret measurements of the evaporation of semi-volatile organic components from binary aqueous/organic aerosol and the hygroscopic growth of involatile inorganic aerosol. The measurements have been made by the aerosol optical tweezers technique, which allows the simultaneous retrieval of particle size and refractive index with high accuracy. We suggest that this approach will be particularly valuable for investigating the thermodynamic behaviour of mixed component aqueous aerosol and will allow the accurate derivation of solution phase equilibrium properties that are prone to large uncertainties when measurements are made simply of the change in particle size with gas phase relative humidity.
DOI
We present a new approach to study the equilibrium gas-particle partitioning of volatile and semi-volatile organic components in aqueous aerosol, deriving a correlational analysis method that examines and interprets simultaneous and correlated fluctuations in particle size and composition. From this approach, changes in particle size driven by organic component evaporation can be clearly resolved from size changes driven by hygroscopicity and fluctuations in environmental conditions. The approach is used to interpret measurements of the evaporation of semi-volatile organic components from binary aqueous/organic aerosol and the hygroscopic growth of involatile inorganic aerosol. The measurements have been made by the aerosol optical tweezers technique, which allows the simultaneous retrieval of particle size and refractive index with high accuracy. We suggest that this approach will be particularly valuable for investigating the thermodynamic behaviour of mixed component aqueous aerosol and will allow the accurate derivation of solution phase equilibrium properties that are prone to large uncertainties when measurements are made simply of the change in particle size with gas phase relative humidity.
DOI
Sorting Nanoparticles with Intertwined Plasmonic and Thermo-Hydrodynamical Forces
A. Cuche, A. Canaguier-Durand, E. Devaux, J. A. Hutchison, C. Genet, and T. W. Ebbesen
We exploit plasmonic and thermo-hydrodynamical forces to sort gold nanoparticles in a microfluidic environment. In the appropriate regime, the experimental data extracted from a Brownian statistical analysis of the kinetic motions are in good agreement with Mie-type theoretical evaluations of the optical forces acting on the nanoparticles in the plasmonic near field. This analysis enables us to demonstrate the importance of thermal and hydrodynamical effects in a sorting perspective.
DOI
We exploit plasmonic and thermo-hydrodynamical forces to sort gold nanoparticles in a microfluidic environment. In the appropriate regime, the experimental data extracted from a Brownian statistical analysis of the kinetic motions are in good agreement with Mie-type theoretical evaluations of the optical forces acting on the nanoparticles in the plasmonic near field. This analysis enables us to demonstrate the importance of thermal and hydrodynamical effects in a sorting perspective.
DOI
The bifoil photodyne: a photonic crystal oscillator
J. E. Lugo, R. Doti, N. Sanchez, M. B. de la Mora, J. A. del Rio & J. Faubert
Optical tweezers is an example how to use light to generate a physical force. They have been used to levitate viruses, bacteria, cells, and sub cellular organisms. Nonetheless it would be beneficial to use such force to develop a new kind of applications. However the radiation pressure usually is small to think in moving larger objects. Currently, there is some research investigating novel photonic working principles to generate a higher force. Here, we studied theoretically and experimentally the induction of electromagnetic forces in one-dimensional photonic crystals when light impinges on the off-axis direction. The photonic structure consists of a micro-cavity like structure formed of two one-dimensional photonic crystals made of free-standing porous silicon, separated by a variable air gap and the working wavelength is 633 nm. We show experimental evidence of this force when the photonic structure is capable of making auto-oscillations and forced-oscillations. We measured peak displacements and velocities ranging from 2 up to 35 microns and 0.4 up to 2.1 mm/s with a power of 13 mW. Recent evidence showed that giant resonant light forces could induce average velocity values of 0.45 mm/s in microspheres embedded in water with 43 mW light power.
DOI
Optical tweezers is an example how to use light to generate a physical force. They have been used to levitate viruses, bacteria, cells, and sub cellular organisms. Nonetheless it would be beneficial to use such force to develop a new kind of applications. However the radiation pressure usually is small to think in moving larger objects. Currently, there is some research investigating novel photonic working principles to generate a higher force. Here, we studied theoretically and experimentally the induction of electromagnetic forces in one-dimensional photonic crystals when light impinges on the off-axis direction. The photonic structure consists of a micro-cavity like structure formed of two one-dimensional photonic crystals made of free-standing porous silicon, separated by a variable air gap and the working wavelength is 633 nm. We show experimental evidence of this force when the photonic structure is capable of making auto-oscillations and forced-oscillations. We measured peak displacements and velocities ranging from 2 up to 35 microns and 0.4 up to 2.1 mm/s with a power of 13 mW. Recent evidence showed that giant resonant light forces could induce average velocity values of 0.45 mm/s in microspheres embedded in water with 43 mW light power.
DOI
Surface charge and hydrodynamic coefficient measurements ofBacillus subtilis spore by Optical Tweezers
Giuseppe Pesce, Giulia Rusciano, Antonio Sasso, Rachele Isticato, Teja Sirec, Ezio Ricca
In this work we report on the simultaneous measurement of the hydrodynamic coefficient and the electric charge of single Bacillus subtilis spores. The latter has great importance in protein binding to spores and in the adhesion of spores onto surfaces. The charge and the hydrodynamic coefficient were measured by an accurate procedure based on the analysis of the motion of single spores confined by an optical trap. The technique has been validated using charged spherical polystyrene beads. The excellent agreement of our results with the expected values demonstrates the quality of our procedure. We measured the charge of spores of B. subtilis purified from a wild type strain and from two isogenic mutants characterized by an altered spore surface. Our technique is able to discriminate the three spore types used, by their charge and by their hydrodynamic coefficient which is related to the hydrophobic properties of the spore surface.
DOI
In this work we report on the simultaneous measurement of the hydrodynamic coefficient and the electric charge of single Bacillus subtilis spores. The latter has great importance in protein binding to spores and in the adhesion of spores onto surfaces. The charge and the hydrodynamic coefficient were measured by an accurate procedure based on the analysis of the motion of single spores confined by an optical trap. The technique has been validated using charged spherical polystyrene beads. The excellent agreement of our results with the expected values demonstrates the quality of our procedure. We measured the charge of spores of B. subtilis purified from a wild type strain and from two isogenic mutants characterized by an altered spore surface. Our technique is able to discriminate the three spore types used, by their charge and by their hydrodynamic coefficient which is related to the hydrophobic properties of the spore surface.
DOI
Quantitative characterization for dielectrophoretic behavior of biological cells using optical tweezers
In Soo Park, Se Hee Park, Sang Woo Lee, Dae Sung Yoon and Beop-Min Kim
We report a method to precisely quantify dielectrophoretic (DEP) forces and cutoff frequencies (f c) of viable and nonviable yeast cells. The method consists of a two-step process in which generated DEP forces act upon a cell through a micro-electrode device, followed by direct measurement of DEP forces using optical tweezers. DEP behaviors of viable and nonviable yeast cells are monitored as a function of AC frequency. We believe that the proposed method can be used as a powerful platform for cell-based assays to characterize the DEP behavior of various cell types including cancer and normal cells.
DOI
We report a method to precisely quantify dielectrophoretic (DEP) forces and cutoff frequencies (f c) of viable and nonviable yeast cells. The method consists of a two-step process in which generated DEP forces act upon a cell through a micro-electrode device, followed by direct measurement of DEP forces using optical tweezers. DEP behaviors of viable and nonviable yeast cells are monitored as a function of AC frequency. We believe that the proposed method can be used as a powerful platform for cell-based assays to characterize the DEP behavior of various cell types including cancer and normal cells.
DOI
Subdiffraction-Limited Quantum Imaging within a Living Cell
Michael A. Taylor, Jiri Janousek, Vincent Daria, Joachim Knittel, Boris Hage, Hans-A. Bachor and Warwick P. Bowen
We report both subdiffraction-limited quantum metrology and quantum-enhanced spatial resolution for the first time in a biological context. Nanoparticles are tracked with quantum-correlated light as they diffuse through an extended region of a living cell in a quantum-enhanced photonic-force microscope. This allows spatial structure within the cell to be mapped at length scales down to 10 nm. Control experiments in water show a 14% resolution enhancement compared to experiments with coherent light. Our results confirm the long-standing prediction that quantum-correlated light can enhance spatial resolution at the nanoscale and in biology. Combined with state-of-the-art quantum light sources, this technique provides a path towards an order of magnitude improvement in resolution over similar classical imaging techniques.
DOI
We report both subdiffraction-limited quantum metrology and quantum-enhanced spatial resolution for the first time in a biological context. Nanoparticles are tracked with quantum-correlated light as they diffuse through an extended region of a living cell in a quantum-enhanced photonic-force microscope. This allows spatial structure within the cell to be mapped at length scales down to 10 nm. Control experiments in water show a 14% resolution enhancement compared to experiments with coherent light. Our results confirm the long-standing prediction that quantum-correlated light can enhance spatial resolution at the nanoscale and in biology. Combined with state-of-the-art quantum light sources, this technique provides a path towards an order of magnitude improvement in resolution over similar classical imaging techniques.
DOI
Monday, February 17, 2014
Proof-of-principle for simple microshelter-assisted buffer exchange in laser tweezers: interaction of Hypericin with single cells
Moktar Amhmed Omar, Pavol Miskovsky and Gregor Bano
Microshelters (i.e. thin dead-end side-arms of fluid channels) are used to aid buffer exchange in optical tweezers experiments. The basic idea is to transfer trapped objects into microshelters during the buffer exchange process. Particles “hidden” in microshelters become insensitive to extreme flow conditions in the main fluid channel, which minimizes the requirements for the applied flow system. The construction scheme of a simple microshelter system is described. The concept has been tested by fluorescence measurements on Hypericin interaction with trapped yeast cells in different environments.
DOI
Microshelters (i.e. thin dead-end side-arms of fluid channels) are used to aid buffer exchange in optical tweezers experiments. The basic idea is to transfer trapped objects into microshelters during the buffer exchange process. Particles “hidden” in microshelters become insensitive to extreme flow conditions in the main fluid channel, which minimizes the requirements for the applied flow system. The construction scheme of a simple microshelter system is described. The concept has been tested by fluorescence measurements on Hypericin interaction with trapped yeast cells in different environments.
DOI
Three-dimensional analysis of optical forces generated by an active tractor beam using radial polarization
Luis Carretero, Pablo Acebal, and Salvador Blaya
We theoretically study the three-dimensional behavior of nanoparticles in an active optical conveyor. To do this, we solved the Langevin equation when the forces are generated by a focusing system at the near field. Analytical expressions for the optical forces generated by the optical conveyor were obtained by solving the Richards and Wolf vectorial diffraction integrals in an approximated form when a mask of two annular pupils is illuminated by a radially polarized Hermite-Gauss beam. Trajectories, in both the transverse plane and the longitudinal direction, are analyzed showing that the behavior of the optical conveyor can be optimized by conveniently choosing the configuration of the mask of the two annular pupils (inner and outer radius of the two rings) in order to trap and transport all particles at the focal plane.
DOI
We theoretically study the three-dimensional behavior of nanoparticles in an active optical conveyor. To do this, we solved the Langevin equation when the forces are generated by a focusing system at the near field. Analytical expressions for the optical forces generated by the optical conveyor were obtained by solving the Richards and Wolf vectorial diffraction integrals in an approximated form when a mask of two annular pupils is illuminated by a radially polarized Hermite-Gauss beam. Trajectories, in both the transverse plane and the longitudinal direction, are analyzed showing that the behavior of the optical conveyor can be optimized by conveniently choosing the configuration of the mask of the two annular pupils (inner and outer radius of the two rings) in order to trap and transport all particles at the focal plane.
DOI
Brownian Motion in a Speckle Light Field: Tunable Anomalous Diffusion and Selective Optical Manipulation
Giorgio Volpe, Giovanni Volpe & Sylvain Gigan
The motion of particles in random potentials occurs in several natural phenomena ranging from the mobility of organelles within a biological cell to the diffusion of stars within a galaxy. A Brownian particle moving in the random optical potential associated to a speckle pattern, i.e., a complex interference pattern generated by the scattering of coherent light by a random medium, provides an ideal model system to study such phenomena. Here, we derive a theory for the motion of a Brownian particle in a speckle field and, in particular, we identify its universal characteristic timescale. Based on this theoretical insight, we show how speckle light fields can be used to control the anomalous diffusion of a Brownian particle and to perform some basic optical manipulation tasks such as guiding and sorting. Our results might broaden the perspectives of optical manipulation for real-life applications.
DOI
The motion of particles in random potentials occurs in several natural phenomena ranging from the mobility of organelles within a biological cell to the diffusion of stars within a galaxy. A Brownian particle moving in the random optical potential associated to a speckle pattern, i.e., a complex interference pattern generated by the scattering of coherent light by a random medium, provides an ideal model system to study such phenomena. Here, we derive a theory for the motion of a Brownian particle in a speckle field and, in particular, we identify its universal characteristic timescale. Based on this theoretical insight, we show how speckle light fields can be used to control the anomalous diffusion of a Brownian particle and to perform some basic optical manipulation tasks such as guiding and sorting. Our results might broaden the perspectives of optical manipulation for real-life applications.
DOI
Controllable orientation of single silver nanowire using two fiber probes
Xiaohao Xu, Chang Cheng, Hongbao Xin, Hongxiang Lei & Baojun Li
We report a strategy for realizing precise orientation of single silver nanowire using two fiber probes. By launching a laser of 980 nm wavelength into the two fibers, single silver nanowire with a diameter of 600 nm and a length of 6.5 μm suspended in water was trapped and rotated by optical torque resulting from its interaction with optical fields outputted from the fiber probes. Angular orientation of the nanowire was controlled by varying the relative distance between the two fiber probes. The angular stiffness, which refers to the stability of orientation, was estimated to be on the order of 10−19 J/rad2·mW. The experiments were interpreted by theoretical analysis.
DOI
We report a strategy for realizing precise orientation of single silver nanowire using two fiber probes. By launching a laser of 980 nm wavelength into the two fibers, single silver nanowire with a diameter of 600 nm and a length of 6.5 μm suspended in water was trapped and rotated by optical torque resulting from its interaction with optical fields outputted from the fiber probes. Angular orientation of the nanowire was controlled by varying the relative distance between the two fiber probes. The angular stiffness, which refers to the stability of orientation, was estimated to be on the order of 10−19 J/rad2·mW. The experiments were interpreted by theoretical analysis.
DOI
Loop 2 of myosin is a force-dependent inhibitor of the rigor bond
Amy M. Clobes, William H. Guilford
Myosin’s actin-binding loop (loop 2) carries a charge opposite to that of its binding site on actin and is thought to play an important role in ionic interactions between the two molecules during the initial binding step. However, no subsequent role has been identified for loop 2 in actin-myosin binding. We used an optical trap to measure bond formation and bond rupture between actin and rigor heavy meromyosin when loaded perpendicular to the filament axis. We studied HMM with intact or proteolytically cleaved loop 2 at low and physiologic ionic strength. Here we show that the presence of intact loop 2 allows actomyosin bonds to form quickly and that they do so in a short-lived bound state. Increasing tensile load causes the transition to a long-lived state—the distinguishing behavior of a catch bond. When loop 2 was cleaved catch bond behavior was abrogated leaving only a long-lived state. These data suggest that in addition to its role in locating binding sites on actin, loop 2 is also a force-dependent inhibitor of the long-lived actomyosin complex. This may be important for reducing the duty ratio and increasing the shortening velocity of actomyosin at low forces.
DOI
Myosin’s actin-binding loop (loop 2) carries a charge opposite to that of its binding site on actin and is thought to play an important role in ionic interactions between the two molecules during the initial binding step. However, no subsequent role has been identified for loop 2 in actin-myosin binding. We used an optical trap to measure bond formation and bond rupture between actin and rigor heavy meromyosin when loaded perpendicular to the filament axis. We studied HMM with intact or proteolytically cleaved loop 2 at low and physiologic ionic strength. Here we show that the presence of intact loop 2 allows actomyosin bonds to form quickly and that they do so in a short-lived bound state. Increasing tensile load causes the transition to a long-lived state—the distinguishing behavior of a catch bond. When loop 2 was cleaved catch bond behavior was abrogated leaving only a long-lived state. These data suggest that in addition to its role in locating binding sites on actin, loop 2 is also a force-dependent inhibitor of the long-lived actomyosin complex. This may be important for reducing the duty ratio and increasing the shortening velocity of actomyosin at low forces.
DOI
Monday, February 10, 2014
Spectroscopy, Manipulation and Trapping of Neutral Atoms, Molecules, and Other Particles Using Optical Nanofibers: A Review
Michael J. Morrissey, Kieran Deasy, Mary Frawley, Ravi Kumar, Eugen Prel, Laura Russell, Viet Giang Truong and Síle Nic Chormaic
The use of tapered optical fibers, i.e., optical nanofibers, for spectroscopy and the detection of small numbers of particles, such as neutral atoms or molecules, has been gaining interest in recent years. In this review, we briefly introduce the optical nanofiber, its fabrication, and optical mode propagation within. We discuss recent progress on the integration of optical nanofibers into laser-cooled atom and vapor systems, paying particular attention to spectroscopy, cold atom cloud characterization, and optical trapping schemes. Next, a natural extension of this work to molecules is introduced. Finally, we consider several alternatives to optical nanofibers that display some advantages for specific applications.
DOI
The use of tapered optical fibers, i.e., optical nanofibers, for spectroscopy and the detection of small numbers of particles, such as neutral atoms or molecules, has been gaining interest in recent years. In this review, we briefly introduce the optical nanofiber, its fabrication, and optical mode propagation within. We discuss recent progress on the integration of optical nanofibers into laser-cooled atom and vapor systems, paying particular attention to spectroscopy, cold atom cloud characterization, and optical trapping schemes. Next, a natural extension of this work to molecules is introduced. Finally, we consider several alternatives to optical nanofibers that display some advantages for specific applications.
DOI
Combining temperature and force to study folding of an RNA hairpin
William Stephenson, Sean Keller, Rachel Santiago, James E. Albrecht, Papa Nii Asare-Okai, Scott A. Tenenbaum, Michael Zuker and Pan T. X. Li
RNA folding in cells typically occurs at mesophilic temperatures. However, in vitro, RNA can be unfolded either by increasing temperature to values that are much higher than physiological, or by mechanically pulling structures apart at ambient temperature. To directly study RNA folding at physiological temperatures and to unify thermodynamics measured by melting and pulling, we developed temperature-controlled optical tweezers (thermal tweezers) that can be used to mechanically unfold single RNA molecules at mesophilic temperatures. Folding of a 20-base-pair tetraloop hairpin was studied under different ionic conditions and at temperatures ranging from 22 °C to 42 °C. At each temperature, single hairpin molecules were held at constant force, and their two-state folding equilibria were monitored. The change in free energy derived from these measurements was used to construct a phase diagram of RNA structure using force and temperature as variables. Furthermore, we derived ΔG0pN,T, the folding free energy at zero force and temperature T, by subtracting the stretching energy of unfolded RNA from the reversible mechanical work done to unfold the hairpin. ΔG0pN,T and its salt dependence agree reasonably well with the predictions by the nearest neighbor model. Under each ionic condition, ΔG0pN,T depended linearly on temperature, yielding ΔHexp and ΔSexp that also matched the predictions. The combination of force and temperature to study RNA folding is a step toward unifying thermodynamics measured by thermal melting and mechanical unfolding, and opens a new path for directly monitoring temperature induced RNA structural changes, as it occurs often in biology.
DOI
RNA folding in cells typically occurs at mesophilic temperatures. However, in vitro, RNA can be unfolded either by increasing temperature to values that are much higher than physiological, or by mechanically pulling structures apart at ambient temperature. To directly study RNA folding at physiological temperatures and to unify thermodynamics measured by melting and pulling, we developed temperature-controlled optical tweezers (thermal tweezers) that can be used to mechanically unfold single RNA molecules at mesophilic temperatures. Folding of a 20-base-pair tetraloop hairpin was studied under different ionic conditions and at temperatures ranging from 22 °C to 42 °C. At each temperature, single hairpin molecules were held at constant force, and their two-state folding equilibria were monitored. The change in free energy derived from these measurements was used to construct a phase diagram of RNA structure using force and temperature as variables. Furthermore, we derived ΔG0pN,T, the folding free energy at zero force and temperature T, by subtracting the stretching energy of unfolded RNA from the reversible mechanical work done to unfold the hairpin. ΔG0pN,T and its salt dependence agree reasonably well with the predictions by the nearest neighbor model. Under each ionic condition, ΔG0pN,T depended linearly on temperature, yielding ΔHexp and ΔSexp that also matched the predictions. The combination of force and temperature to study RNA folding is a step toward unifying thermodynamics measured by thermal melting and mechanical unfolding, and opens a new path for directly monitoring temperature induced RNA structural changes, as it occurs often in biology.
DOI
Aerosol droplet optical trap loading using surface acoustic wave nebulization
S. Anand, J. Nylk, S. L. Neale, C. Dodds, S. Grant, M. H. Ismail, J. Reboud, J. M. Cooper, and D. McGloin
We demonstrate the use of surface acoustic wave nebulization (SAWN) to load optical traps. We show that the droplets sizes produced can be tuned by altering the RF frequency applied to the devices, which leads to more control over the sizes of trapped particles. Typically the size distribution of the liquid aerosols delivered using SAWN is smaller than via a standard commercial nebulization device. The ability to trap a range of liquids or small solid particles, not readily accessible using other ultrasonic devices, is also demonstrated both in optical tweezers and dual beam fiber traps.
DOI
We demonstrate the use of surface acoustic wave nebulization (SAWN) to load optical traps. We show that the droplets sizes produced can be tuned by altering the RF frequency applied to the devices, which leads to more control over the sizes of trapped particles. Typically the size distribution of the liquid aerosols delivered using SAWN is smaller than via a standard commercial nebulization device. The ability to trap a range of liquids or small solid particles, not readily accessible using other ultrasonic devices, is also demonstrated both in optical tweezers and dual beam fiber traps.
DOI
Dressing plasmon resonance with particle-microcavity architecture for efficient nano-optical trapping and sensing
Haixi Zhang, Yanyan Zhou, Xia Yu, Feng Luan, Jianbin Xu, Hock-Chun Ong, and Ho-Pui Ho
We propose a particle-microcavity scheme for efficient optical trapping and sensing. When a resonant plasmonic nanoparticle (NP) is placed inside a microcavity with high Q-factor, sensitivity is enhanced in the far-field extinction while near-field around the NP is barely affected. Stable near-field and high sensitivity for optical trapping and ultrasensitive detection of nanosized targets are therefore realized simultaneously. Such a particle-microcavity system opens up a new hybrid nanophotonic device platform that combines the unique merits of conventional and plasmonic integrated photonics.
DOI
We propose a particle-microcavity scheme for efficient optical trapping and sensing. When a resonant plasmonic nanoparticle (NP) is placed inside a microcavity with high Q-factor, sensitivity is enhanced in the far-field extinction while near-field around the NP is barely affected. Stable near-field and high sensitivity for optical trapping and ultrasensitive detection of nanosized targets are therefore realized simultaneously. Such a particle-microcavity system opens up a new hybrid nanophotonic device platform that combines the unique merits of conventional and plasmonic integrated photonics.
DOI
Tuesday, February 4, 2014
In situ Quantification of Ammonium Sulfate in Single Aerosol Droplets by Means of Laser Trapping and Raman Spectroscopy
Ishizaka S, Yamauchi K, Kitamura N.
Noncontact levitation in air of single micrometer-sized water droplets containing ammonium sulfate was successful by a laser trapping technique. The trapping laser beam was also used simultaneously as an excitation light source for the Raman spectroscopy of trapped droplets. Raman scattering of the symmetric stretching vibration of the SO42− anion and the OH stretching vibrations of H2O were observed at 980 and 3420 cm−1, respectively. The intensity ratio of these two Raman peaks was linearly proportional to the ammonium sulfate concentration in water. Therefore, the in situ quantification of ammonium sulfate in single aerosol droplets was achieved by means of laser trapping and Raman spectroscopy. To the best of our knowledge, this study is the first experimental observation of the independence of ammonium sulfate concentrations of aerosol water droplets to those of the mother solutions under constant relative humidity conditions.
DOI
Noncontact levitation in air of single micrometer-sized water droplets containing ammonium sulfate was successful by a laser trapping technique. The trapping laser beam was also used simultaneously as an excitation light source for the Raman spectroscopy of trapped droplets. Raman scattering of the symmetric stretching vibration of the SO42− anion and the OH stretching vibrations of H2O were observed at 980 and 3420 cm−1, respectively. The intensity ratio of these two Raman peaks was linearly proportional to the ammonium sulfate concentration in water. Therefore, the in situ quantification of ammonium sulfate in single aerosol droplets was achieved by means of laser trapping and Raman spectroscopy. To the best of our knowledge, this study is the first experimental observation of the independence of ammonium sulfate concentrations of aerosol water droplets to those of the mother solutions under constant relative humidity conditions.
DOI
Sequence-dependent base pair stepping dynamics in XPD helicase unwinding
Zhi Qi, Robert A Pugh, Maria Spies, Yann R Chemla
Helicases couple the chemical energy of ATP hydrolysis to directional translocation along nucleic acids and transient duplex separation. Understanding helicase mechanism requires that the basic physicochemical process of base pair separation be understood. This necessitates monitoring helicase activity directly, at high spatio-temporal resolution. Using optical tweezers with single base pair (bp) resolution, we analyzed DNA unwinding by XPD helicase, a Superfamily 2 (SF2) DNA helicase involved in DNA repair and transcription initiation. We show that monomeric XPD unwinds duplex DNA in 1-bp steps, yet exhibits frequent backsteps and undergoes conformational transitions manifested in 5-bp backward and forward steps. Quantifying the sequence dependence of XPD stepping dynamics with near base pair resolution, we provide the strongest and most direct evidence thus far that forward, single-base pair stepping of a helicase utilizes the spontaneous opening of the duplex. The proposed unwinding mechanism may be a universal feature of DNA helicases that move along DNA phosphodiester backbones.
DOI
Helicases couple the chemical energy of ATP hydrolysis to directional translocation along nucleic acids and transient duplex separation. Understanding helicase mechanism requires that the basic physicochemical process of base pair separation be understood. This necessitates monitoring helicase activity directly, at high spatio-temporal resolution. Using optical tweezers with single base pair (bp) resolution, we analyzed DNA unwinding by XPD helicase, a Superfamily 2 (SF2) DNA helicase involved in DNA repair and transcription initiation. We show that monomeric XPD unwinds duplex DNA in 1-bp steps, yet exhibits frequent backsteps and undergoes conformational transitions manifested in 5-bp backward and forward steps. Quantifying the sequence dependence of XPD stepping dynamics with near base pair resolution, we provide the strongest and most direct evidence thus far that forward, single-base pair stepping of a helicase utilizes the spontaneous opening of the duplex. The proposed unwinding mechanism may be a universal feature of DNA helicases that move along DNA phosphodiester backbones.
DOI
Discriminatory optical force for chiral molecules
Robert P Cameron, Stephen M Barnett and Alison M Yao
We suggest that the force F exerted upon a chiral molecule by light assumes the form $\mathbf {F}=a\boldsymbol {\nabla } w+b\boldsymbol {\nabla } h$ under appropriate circumstances, where a and b pertain to the molecule whilst w and h are the local densities of electric energy and helicity in the optical field; the gradients $\boldsymbol {\nabla }$ of these quantities thus governing the molecule's centre-of-mass motion. Whereas a is identical for the mirror-image forms or enantiomers of the molecule, b has opposite signs; the associated contribution to F therefore pointing in opposite directions. A simple optical field is presented for which $\boldsymbol {\nabla } w$ vanishes but $\boldsymbol {\nabla }h$ does not, so that F is absolutely discriminatory. We then present two potential applications: a Stern–Gerlach-type deflector capable of spatially separating the enantiomers of a chiral molecule and a diffraction grating to which chiral molecules alone are sensitive; the resulting diffraction patterns thus encoding information about their chiral geometry.
DOI
We suggest that the force F exerted upon a chiral molecule by light assumes the form $\mathbf {F}=a\boldsymbol {\nabla } w+b\boldsymbol {\nabla } h$ under appropriate circumstances, where a and b pertain to the molecule whilst w and h are the local densities of electric energy and helicity in the optical field; the gradients $\boldsymbol {\nabla }$ of these quantities thus governing the molecule's centre-of-mass motion. Whereas a is identical for the mirror-image forms or enantiomers of the molecule, b has opposite signs; the associated contribution to F therefore pointing in opposite directions. A simple optical field is presented for which $\boldsymbol {\nabla } w$ vanishes but $\boldsymbol {\nabla }h$ does not, so that F is absolutely discriminatory. We then present two potential applications: a Stern–Gerlach-type deflector capable of spatially separating the enantiomers of a chiral molecule and a diffraction grating to which chiral molecules alone are sensitive; the resulting diffraction patterns thus encoding information about their chiral geometry.
DOI
Membrane Shape Modulates Transmembrane Protein Distribution
Sophie Aimon, Andrew Callan-Jones, Alice Berthaud, Mathieu Pinot, Gilman E.S. Toombes, Patricia Bassereau
Although membrane shape varies greatly throughout the cell, the contribution of membrane curvature to transmembrane protein targeting is unknown because of the numerous sorting mechanisms that take place concurrently in cells. To isolate the effect of membrane shape, we used cell-sized giant unilamellar vesicles (GUVs) containing either the potassium channel KvAP or the water channel AQP0 to form membrane nanotubes with controlled radii. Whereas the AQP0 concentrations in flat and curved membranes were indistinguishable, KvAP was enriched in the tubes, with greater enrichment in more highly curved membranes. Fluorescence recovery after photobleaching measurements showed that both proteins could freely diffuse through the neck between the tube and GUV, and the effect of each protein on membrane shape and stiffness was characterized using a thermodynamic sorting model. This study establishes the importance of membrane shape for targeting transmembrane proteins and provides a method for determining the effective shape and flexibility of membrane proteins.
DOI
Although membrane shape varies greatly throughout the cell, the contribution of membrane curvature to transmembrane protein targeting is unknown because of the numerous sorting mechanisms that take place concurrently in cells. To isolate the effect of membrane shape, we used cell-sized giant unilamellar vesicles (GUVs) containing either the potassium channel KvAP or the water channel AQP0 to form membrane nanotubes with controlled radii. Whereas the AQP0 concentrations in flat and curved membranes were indistinguishable, KvAP was enriched in the tubes, with greater enrichment in more highly curved membranes. Fluorescence recovery after photobleaching measurements showed that both proteins could freely diffuse through the neck between the tube and GUV, and the effect of each protein on membrane shape and stiffness was characterized using a thermodynamic sorting model. This study establishes the importance of membrane shape for targeting transmembrane proteins and provides a method for determining the effective shape and flexibility of membrane proteins.
DOI
Monday, February 3, 2014
Mobility Analysis of Super-Resolved Proteins on Optically Stretched DNA: Comparing Imaging Techniques and Parameters
Dr. Iddo Heller, Gerrit Sitters, Onno D. Broekmans, Dr. Andreas S. Biebricher, Prof. Dr. Gijs J. L. Wuite, Prof. Dr. Erwin J. G. Peterman
Fluorescence microscopy in conjunction with optical tweezers is well suited to the study of protein mobility on DNA. Here, we evaluate the benefits and drawbacks of super-resolution and conventional imaging techniques for the analysis of one-dimensional (1D) protein diffusion as commonly observed for DNA-binding proteins. In particular, we demonstrate the visualization of DNA-bound proteins using wide-field, confocal, and stimulated emission depletion (STED) microscopy. We review the suitability of these techniques to conditions of high protein density, and quantify their performance in terms of spatial and temporal resolution. Tracking proteins on DNA forces one to make a choice between localization precision on the one hand, and the number and rate of localizations on the other, by altering imaging modality, excitation intensity, and acquisition rate. Using simulated diffusion data, we quantify the effect of these imaging conditions on the accuracy of 1D diffusion analysis. In addition, we consider the case of diffusion confined between local roadblocks, a case particularly relevant for proteins bound to DNA. Together these results provide guidelines that can assist in judiciously optimizing the experimental conditions required for the analysis of protein mobility on DNA and other 1D systems.
DOI
Fluorescence microscopy in conjunction with optical tweezers is well suited to the study of protein mobility on DNA. Here, we evaluate the benefits and drawbacks of super-resolution and conventional imaging techniques for the analysis of one-dimensional (1D) protein diffusion as commonly observed for DNA-binding proteins. In particular, we demonstrate the visualization of DNA-bound proteins using wide-field, confocal, and stimulated emission depletion (STED) microscopy. We review the suitability of these techniques to conditions of high protein density, and quantify their performance in terms of spatial and temporal resolution. Tracking proteins on DNA forces one to make a choice between localization precision on the one hand, and the number and rate of localizations on the other, by altering imaging modality, excitation intensity, and acquisition rate. Using simulated diffusion data, we quantify the effect of these imaging conditions on the accuracy of 1D diffusion analysis. In addition, we consider the case of diffusion confined between local roadblocks, a case particularly relevant for proteins bound to DNA. Together these results provide guidelines that can assist in judiciously optimizing the experimental conditions required for the analysis of protein mobility on DNA and other 1D systems.
DOI
Single Cell Transfection with Single Molecule Resolution Using a Synthetic Nanopore
Volker Kurz , Tetsuya Tanaka , and Gregory Timp
We report the development of a single cell gene delivery system based on electroporation using a synthetic nanopore, that is not only highly specific and very efficient, but also transfects with single molecule resolution at low voltage (1V) with minimal perturbation to the cell. Such a system can be used to control gene expression with unprecedented precision—no other method offers such capabilities.
DOI
We report the development of a single cell gene delivery system based on electroporation using a synthetic nanopore, that is not only highly specific and very efficient, but also transfects with single molecule resolution at low voltage (1V) with minimal perturbation to the cell. Such a system can be used to control gene expression with unprecedented precision—no other method offers such capabilities.
DOI
A vertebrate myosin-I structure reveals unique insights into myosin mechanochemical tuning
Henry Shuman, Michael J. Greenberg, Adam Zwolak, Tianming Lin, Charles V. Sindelar, Roberto Dominguez, and E. Michael Ostap
Myosins are molecular motors that power diverse cellular processes, such as rapid organelle transport, muscle contraction, and tension-sensitive anchoring. The structural adaptations in the motor that allow for this functional diversity are not known, due, in part, to the lack of high-resolution structures of highly tension-sensitive myosins. We determined a 2.3-Å resolution structure of apo-myosin-Ib (Myo1b), which is the most tension-sensitive myosin characterized. We identified a striking unique orientation of structural elements that position the motor’s lever arm. This orientation results in a cavity between the motor and lever arm that holds a 10-residue stretch of N-terminal amino acids, a region that is divergent among myosins. Single-molecule and biochemical analyses show that the N terminus plays an important role in stabilizing the post power-stroke conformation of Myo1b and in tuning the rate of the force-sensitive transition. We propose that this region plays a general role in tuning the mechanochemical properties of myosins.
Myosins are molecular motors that power diverse cellular processes, such as rapid organelle transport, muscle contraction, and tension-sensitive anchoring. The structural adaptations in the motor that allow for this functional diversity are not known, due, in part, to the lack of high-resolution structures of highly tension-sensitive myosins. We determined a 2.3-Å resolution structure of apo-myosin-Ib (Myo1b), which is the most tension-sensitive myosin characterized. We identified a striking unique orientation of structural elements that position the motor’s lever arm. This orientation results in a cavity between the motor and lever arm that holds a 10-residue stretch of N-terminal amino acids, a region that is divergent among myosins. Single-molecule and biochemical analyses show that the N terminus plays an important role in stabilizing the post power-stroke conformation of Myo1b and in tuning the rate of the force-sensitive transition. We propose that this region plays a general role in tuning the mechanochemical properties of myosins.
Trap position control in the vicinity of reflecting surfaces in optical tweezers
D. A. Shilkin, E. V. Lyubin, I. V. Soboleva, A. A. Fedyanin
Shift of the trap position from the laser beam waist of optical tweezers is studied experimentally in the presence of a reflecting surface in the vicinity of the focal plane. A standing wave is formed owing to the interference of waves forming the waist and reflected from the surface. The standing wave is shown to affect significantly the resulting trap position. The distance between the surface and the stable optical trap as a function of the trapped particle size is studied numerically. A new method to stabilize the position of the microparticle relative to the surface is proposed. The localization accuracy is determined by the Brownian fluctuations in optical tweezers and is about 10 nm for effective trap stiffness of 4 × 10−5 N/m.
DOI
Shift of the trap position from the laser beam waist of optical tweezers is studied experimentally in the presence of a reflecting surface in the vicinity of the focal plane. A standing wave is formed owing to the interference of waves forming the waist and reflected from the surface. The standing wave is shown to affect significantly the resulting trap position. The distance between the surface and the stable optical trap as a function of the trapped particle size is studied numerically. A new method to stabilize the position of the microparticle relative to the surface is proposed. The localization accuracy is determined by the Brownian fluctuations in optical tweezers and is about 10 nm for effective trap stiffness of 4 × 10−5 N/m.
DOI
Single-Molecule Measurements of the CCR5 mRNA Unfolding Pathways
Michel de Messieres, Jen-Chien Chang, Ashton Trey Belew, Arturas Meskauskas, Jonathan D. Dinman, Arthur La Porta
Secondary or tertiary structure in an mRNA, such as a pseudoknot, can create a physical barrier that requires the ribosome to generate additional force to translocate. The presence of such a barrier can dramatically increase the probability that the ribosome will shift into an alternate reading frame, in which a different set of codons is recognized. The detailed biophysical mechanism by which frameshifting is induced remains unknown. Here we employ optical trapping techniques to investigate the structure of a −1 programmed ribosomal frameshift (−1 PRF) sequence element located in the CCR5 mRNA, which encodes a coreceptor for HIV-1 and is, to our knowledge, the first known human −1 PRF signal of nonviral origin. We begin by presenting a set of computationally predicted structures that include pseudoknots. We then employ what we believe to be new analytical techniques for measuring the effective free energy landscapes of biomolecules. We find that the −1 PRF element manifests several distinct unfolding pathways when subject to end-to-end force, one of which is consistent with a proposed pseudoknot conformation, and another of which we have identified as a folding intermediate. The dynamic ensemble of conformations that CCR5 mRNA exhibits in the single-molecule experiments may be a significant feature of the frameshifting mechanism.
DOI
Secondary or tertiary structure in an mRNA, such as a pseudoknot, can create a physical barrier that requires the ribosome to generate additional force to translocate. The presence of such a barrier can dramatically increase the probability that the ribosome will shift into an alternate reading frame, in which a different set of codons is recognized. The detailed biophysical mechanism by which frameshifting is induced remains unknown. Here we employ optical trapping techniques to investigate the structure of a −1 programmed ribosomal frameshift (−1 PRF) sequence element located in the CCR5 mRNA, which encodes a coreceptor for HIV-1 and is, to our knowledge, the first known human −1 PRF signal of nonviral origin. We begin by presenting a set of computationally predicted structures that include pseudoknots. We then employ what we believe to be new analytical techniques for measuring the effective free energy landscapes of biomolecules. We find that the −1 PRF element manifests several distinct unfolding pathways when subject to end-to-end force, one of which is consistent with a proposed pseudoknot conformation, and another of which we have identified as a folding intermediate. The dynamic ensemble of conformations that CCR5 mRNA exhibits in the single-molecule experiments may be a significant feature of the frameshifting mechanism.
DOI
Optical tweezers reveal force plateau and internal friction in PEG-induced DNA condensation
Heikki Ojala, Gabija Ziedaite, Anders E. Wallin, Dennis H. Bamford, Edward Hæggström
The simplified artificial environments in which highly complex biological systems are studied do not represent the crowded, dense, salty, and dynamic environment inside the living cell. Consequently, it is important to investigate the effect of crowding agents on DNA. We used a dual-trap optical tweezers instrument to perform force spectroscopy experiments at pull speeds ranging from 0.3 to 270 μm/s on single dsDNA molecules in the presence of poly(ethylene glycol) (PEG) and monovalent salt. PEG of sizes 1,500 and 4,000 Da condensed DNA, and force–extension data contained a force plateau at approximately 1 pN. The level of the force plateau increased with increasing pull speed. During slow pulling the dissipated work increased linearly with pull speed. The calculated friction coefficient did not depend on amount of DNA incorporated in the condensate, indicating internal friction is independent of the condensate size. PEG300 had no effect on the dsDNA force–extension curve. The force plateau implies that condensation induced by crowding agents resembles condensation induced by multivalent cations.
DOI
The simplified artificial environments in which highly complex biological systems are studied do not represent the crowded, dense, salty, and dynamic environment inside the living cell. Consequently, it is important to investigate the effect of crowding agents on DNA. We used a dual-trap optical tweezers instrument to perform force spectroscopy experiments at pull speeds ranging from 0.3 to 270 μm/s on single dsDNA molecules in the presence of poly(ethylene glycol) (PEG) and monovalent salt. PEG of sizes 1,500 and 4,000 Da condensed DNA, and force–extension data contained a force plateau at approximately 1 pN. The level of the force plateau increased with increasing pull speed. During slow pulling the dissipated work increased linearly with pull speed. The calculated friction coefficient did not depend on amount of DNA incorporated in the condensate, indicating internal friction is independent of the condensate size. PEG300 had no effect on the dsDNA force–extension curve. The force plateau implies that condensation induced by crowding agents resembles condensation induced by multivalent cations.
DOI
Diverse Metastable Structures Formed by Small Oligomers of α-Synuclein Probed by Force Spectroscopy
Krishna Neupane, Allison Solanki, Iveta Sosova, Miro Belov, Michael T. Woodside
Oligomeric aggregates are widely suspected as toxic agents in diseases caused by protein aggregation, yet they remain poorly characterized, partly because they are challenging to isolate from a heterogeneous mixture of species. We developed an assay for characterizing structure, stability, and kinetics of individual oligomers at high resolution and sensitivity using single-molecule force spectroscopy, and applied it to observe the formation of transient structured aggregates within single oligomers of α-synuclein, an intrinsically-disordered protein linked to Parkinson’s disease. Measurements of the molecular extension as the proteins unfolded under tension in optical tweezers revealed that even small oligomers could form numerous metastable structures, with a surprisingly broad range of sizes. Comparing the structures formed in monomers, dimers and tetramers, we found that the average mechanical stability increased with oligomer size. Most structures formed within a minute, with size-dependent rates. These results provide a new window onto the complex α-synuclein aggregation landscape, characterizing the microscopic structural heterogeneity and kinetics of different pathways.
DOI
Oligomeric aggregates are widely suspected as toxic agents in diseases caused by protein aggregation, yet they remain poorly characterized, partly because they are challenging to isolate from a heterogeneous mixture of species. We developed an assay for characterizing structure, stability, and kinetics of individual oligomers at high resolution and sensitivity using single-molecule force spectroscopy, and applied it to observe the formation of transient structured aggregates within single oligomers of α-synuclein, an intrinsically-disordered protein linked to Parkinson’s disease. Measurements of the molecular extension as the proteins unfolded under tension in optical tweezers revealed that even small oligomers could form numerous metastable structures, with a surprisingly broad range of sizes. Comparing the structures formed in monomers, dimers and tetramers, we found that the average mechanical stability increased with oligomer size. Most structures formed within a minute, with size-dependent rates. These results provide a new window onto the complex α-synuclein aggregation landscape, characterizing the microscopic structural heterogeneity and kinetics of different pathways.
DOI
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