Eric Jaquay, Luis Javier Martínez, Camilo A. Mejia, and Michelle L. Povinelli
We experimentally demonstrate the technique of light-assisted, templated self-assembly (LATS). We excite a guided-resonance mode of a photonic-crystal slab with 1.55 μm laser light to create an array of optical traps. We demonstrate assembly of a square lattice of 520 nm diameter polystyrene particles spaced by 860 nm. Our results demonstrate how LATS can be used to fabricate reconfigurable structures with symmetries different from traditional colloidal self-assembly, which is limited by free energetic constraints.
DOI
Wednesday, July 31, 2013
DNA interaction with Actinomycin D: mechanical measurements reveal the details of the binding data
E. C. Cesconetto, F. S. A. Junior, F. A. P. Crisafuli, O. N. Mesquita, E. B. Ramos and M. S. Rocha
We have studied the interaction between the anticancer drug Actinomycin D (ActD) and the DNA molecule by performing single molecule stretching experiments and atomic force microscopy (AFM) imaging. From the stretching experiments, we determine how the mechanical properties of the DNA–ActD complexes vary as a function of drug concentration, for a fixed DNA concentration. We have found that the persistence lengths of the complexes formed behave non-monotonically: at low concentrations of ActD they are more flexible than the bare DNA molecule and become stiffer at higher concentrations. On the other hand, the contour length increases monotonically as a function of ActD concentration. Using a two-sites quenched disorder statistical model recently developed by us, we were able to extract chemical parameters such as the intrinsic binding constant and the degree of cooperativity from these pure mechanical measurements, thus performing a robust characterization of the interaction. The AFM images, otherwise, were used to measure the bending angle size distribution that ActD introduces on the double-helix structure and the average number of bendings per DNA molecule as a function of drug concentration, two quantities that cannot be determined from the stretching experiments.
We have studied the interaction between the anticancer drug Actinomycin D (ActD) and the DNA molecule by performing single molecule stretching experiments and atomic force microscopy (AFM) imaging. From the stretching experiments, we determine how the mechanical properties of the DNA–ActD complexes vary as a function of drug concentration, for a fixed DNA concentration. We have found that the persistence lengths of the complexes formed behave non-monotonically: at low concentrations of ActD they are more flexible than the bare DNA molecule and become stiffer at higher concentrations. On the other hand, the contour length increases monotonically as a function of ActD concentration. Using a two-sites quenched disorder statistical model recently developed by us, we were able to extract chemical parameters such as the intrinsic binding constant and the degree of cooperativity from these pure mechanical measurements, thus performing a robust characterization of the interaction. The AFM images, otherwise, were used to measure the bending angle size distribution that ActD introduces on the double-helix structure and the average number of bendings per DNA molecule as a function of drug concentration, two quantities that cannot be determined from the stretching experiments.
Holographic Raman Tweezers Controlled by Hand Gestures and Voice Commands
Zoltan Tomori, Marian Antalik, Peter Kesa, Jan Kanka, Petr Jakl, Mojmir Sery, Silvie Bernatova, Pavel Zemanek
Several attempts have appeared recently to control optical trapping systems via touch tablets and cameras instead of a mouse and joystick. Our approach is based on a modern low-cost hardware combined with fingertips and speech recognition software. Positions of operator's hands or fingertips control the positions of trapping beams in holographic optical tweezers that provide optical manipulation with microobjects. We tested and adapted two systems for hands position detection and gestures recognition – Creative Interactive Gesture Camera and Leap Motion. We further enhanced the system of Holographic Raman tweezers (HRT) by voice commands controlling the micropositioning stage and acquisition of Raman spectra. Interface communicates with HRT either directly by which requires adaptation of HRT firmware, or indirectly by simulating mouse and keyboard messages. Its utilization in real experiments speeded up the operator’s communication with the system cca. Two times in comparison with the traditional control by the mouse and the keyboard.
DOI
Several attempts have appeared recently to control optical trapping systems via touch tablets and cameras instead of a mouse and joystick. Our approach is based on a modern low-cost hardware combined with fingertips and speech recognition software. Positions of operator's hands or fingertips control the positions of trapping beams in holographic optical tweezers that provide optical manipulation with microobjects. We tested and adapted two systems for hands position detection and gestures recognition – Creative Interactive Gesture Camera and Leap Motion. We further enhanced the system of Holographic Raman tweezers (HRT) by voice commands controlling the micropositioning stage and acquisition of Raman spectra. Interface communicates with HRT either directly by which requires adaptation of HRT firmware, or indirectly by simulating mouse and keyboard messages. Its utilization in real experiments speeded up the operator’s communication with the system cca. Two times in comparison with the traditional control by the mouse and the keyboard.
DOI
On chip shapeable optical tweezers
C. Renaut, B. Cluzel, J. Dellinger, L. Lalouat, E. Picard, D. Peyrade, E. Hadji & F. de Fornel
Particles manipulation with optical forces is known as optical tweezing. While tweezing in free space with laser beams was established in the 1980s, integrating the optical tweezers on a chip is a challenging task. Recent experiments with plasmonic nanoantennas, microring resonators, and photonic crystal nanocavities have demonstrated optical trapping. However, the optical field of a tweezer made of a single microscopic resonator cannot be shaped. So far, this prevents from optically driven micromanipulations. Here we propose an alternative approach where the shape of the optical trap can be tuned by the wavelength in coupled nanobeam cavities. Using these shapeable tweezers, we present micromanipulation of polystyrene microspheres trapped on a silicon chip. These results show that coupled nanobeam cavities are versatile building blocks for optical near-field engineering. They open the way to much complex integrated tweezers using networks of coupled nanobeam cavities for particles or bio-objects manipulation at a larger scale.
DOI
Particles manipulation with optical forces is known as optical tweezing. While tweezing in free space with laser beams was established in the 1980s, integrating the optical tweezers on a chip is a challenging task. Recent experiments with plasmonic nanoantennas, microring resonators, and photonic crystal nanocavities have demonstrated optical trapping. However, the optical field of a tweezer made of a single microscopic resonator cannot be shaped. So far, this prevents from optically driven micromanipulations. Here we propose an alternative approach where the shape of the optical trap can be tuned by the wavelength in coupled nanobeam cavities. Using these shapeable tweezers, we present micromanipulation of polystyrene microspheres trapped on a silicon chip. These results show that coupled nanobeam cavities are versatile building blocks for optical near-field engineering. They open the way to much complex integrated tweezers using networks of coupled nanobeam cavities for particles or bio-objects manipulation at a larger scale.
DOI
Saturday, July 27, 2013
Partially coherent fluctuating sources that produce the same optical force as a laser beam
J. M. Auñón and M. Nieto-Vesperinas
Inspired by a theory previously derived by Wolf and Collett [Opt. Commun. 25, 293 (1978)], we demonstrate that partially coherent Gaussian–Schell model fluctuating sources (GSMS) produce exactly the same optical forces as a fully coherent laser beam. We also show that this kind of source helps to control the magnitude of the light–matter mechanical interaction in biological samples, which are sensitive to thermal heating induced by higher intensities. The latter is a consequence of the fact that the same photonic force can be obtained with a low-intensity GSMS as with a high-intensity laser beam.
Inspired by a theory previously derived by Wolf and Collett [Opt. Commun. 25, 293 (1978)], we demonstrate that partially coherent Gaussian–Schell model fluctuating sources (GSMS) produce exactly the same optical forces as a fully coherent laser beam. We also show that this kind of source helps to control the magnitude of the light–matter mechanical interaction in biological samples, which are sensitive to thermal heating induced by higher intensities. The latter is a consequence of the fact that the same photonic force can be obtained with a low-intensity GSMS as with a high-intensity laser beam.
Hierarchical Photonic Synthesis of Hybrid Nanoparticle Assemblies
Zijie Yan, Uttam Manna, Wei Qin, Art Camire, Philippe Guyot-Sionnest, and Norbert F. Scherer
Optical “nano-manipulation”, measurement and control of small objects with nanoscale precision, requires strongly localized optical fields that are usually based on user-imposed shaping of the incident optical beam. Here we report an in-situ approach to reshape and enhance electromagnetic (EM) fields using scattering and interference that is concomitant with “dynamic self-assembly” of nanoparticle arrays using simple (unstructured) applied EM fields. We show that Ag nanoparticles (~140 nm dia.) illuminated by coherent light can form linear chains with nanometer precision via strong optical binding interactions. The chains, in turn, create highly shaped EM fields via coherent scattering from the particles, allowing less polarizable particles to be “co-trapped” in both intermediate-scale and near-field regimes. These less polarizable particles include quantum dots (CdSe/ZnS or CdSe/CdZnS core/shell nanocrystals; both are smaller than 10 nm while the latter are further coated by ~ 30 nm thick silica shells) and small Ag nanoparticles (60 nm dia.). This hierarchical optical field induced assembly is a starting point for photonically "building" artificial nanomaterials.
DOI
Optical “nano-manipulation”, measurement and control of small objects with nanoscale precision, requires strongly localized optical fields that are usually based on user-imposed shaping of the incident optical beam. Here we report an in-situ approach to reshape and enhance electromagnetic (EM) fields using scattering and interference that is concomitant with “dynamic self-assembly” of nanoparticle arrays using simple (unstructured) applied EM fields. We show that Ag nanoparticles (~140 nm dia.) illuminated by coherent light can form linear chains with nanometer precision via strong optical binding interactions. The chains, in turn, create highly shaped EM fields via coherent scattering from the particles, allowing less polarizable particles to be “co-trapped” in both intermediate-scale and near-field regimes. These less polarizable particles include quantum dots (CdSe/ZnS or CdSe/CdZnS core/shell nanocrystals; both are smaller than 10 nm while the latter are further coated by ~ 30 nm thick silica shells) and small Ag nanoparticles (60 nm dia.). This hierarchical optical field induced assembly is a starting point for photonically "building" artificial nanomaterials.
DOI
Friday, July 26, 2013
Evanescent wave assisted nanomaterial coating
Samir K. Mondal, Sudipta Sarkar Pal, Dharmadas Kumbhakar, Umesh Tiwari, and Randhir Bhatnagar
In this work we present a novel nanomaterial coating technique using evanescent wave (EW). The gradient force in the EW is used as an optical tweezer for tweezing and self-assembling nanoparticles on the source of EW. As a proof of the concept, we have used a laser coupled etched multimode optical fiber, which generates EW for the EW assisted coating. The section-wise etched multimode optical fiber is horizontally and superficially dipped into a silver/gold nanoparticles solution while the laser is switched on. The fiber is left until the solution recedes due to evaporation leaving the fiber in air. The coating time usually takes 40–50 min at room temperature. The scanning electron microscope image shows uniform and thin coating of self-assembled nanoparticles due to EW around the etched section. A coating thickness <200 nm is achieved. The technique could be useful for making surface-plasmon-resonance-based optical fiber probes and other plasmonic circuits.
DOI
In this work we present a novel nanomaterial coating technique using evanescent wave (EW). The gradient force in the EW is used as an optical tweezer for tweezing and self-assembling nanoparticles on the source of EW. As a proof of the concept, we have used a laser coupled etched multimode optical fiber, which generates EW for the EW assisted coating. The section-wise etched multimode optical fiber is horizontally and superficially dipped into a silver/gold nanoparticles solution while the laser is switched on. The fiber is left until the solution recedes due to evaporation leaving the fiber in air. The coating time usually takes 40–50 min at room temperature. The scanning electron microscope image shows uniform and thin coating of self-assembled nanoparticles due to EW around the etched section. A coating thickness <200 nm is achieved. The technique could be useful for making surface-plasmon-resonance-based optical fiber probes and other plasmonic circuits.
DOI
Tuesday, July 23, 2013
Teamwork in microtubule motors
Roop Mallik, Arpan K. Rai, Pradeep Barak, Ashim Rai, Ambarish Kunwar
Diverse cellular processes are driven by the collective force from multiple motor proteins. Disease-causing mutations cause aberrant function of motors, but the impact is observed at a cellular level and beyond, therefore necessitating an understanding of cell mechanics at the level of motor molecules. One way to do this is by measuring the force generated by ensembles of motors in vivo at single-motor resolution. This has been possible for microtubule motor teams that transport intracellular organelles, revealing unexpected differences between collective and single-molecule function. Here we review how the biophysical properties of single motors, and differences therein, may translate into collective motor function during organelle transport and perhaps in other processes outside transport.
DOI
Diverse cellular processes are driven by the collective force from multiple motor proteins. Disease-causing mutations cause aberrant function of motors, but the impact is observed at a cellular level and beyond, therefore necessitating an understanding of cell mechanics at the level of motor molecules. One way to do this is by measuring the force generated by ensembles of motors in vivo at single-motor resolution. This has been possible for microtubule motor teams that transport intracellular organelles, revealing unexpected differences between collective and single-molecule function. Here we review how the biophysical properties of single motors, and differences therein, may translate into collective motor function during organelle transport and perhaps in other processes outside transport.
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A Novel Single Virus Infection System Reveals That Influenza Virus Preferentially Infects Cells in G1 Phase
Ryuta Ueda, Tadao Sugiura, Shinichiro Kume, Akihiko Ichikawa, Steven Larsen, Hideaki Miyoshi, Hiroaki Hiramatsu, Yasuko Nagatsuka, Fumihito Arai,Yasuo Suzuki, Yoshio Hirabayashi, Toshio Fukuda, Ayae Honda
Influenza virus attaches to sialic acid residues on the surface of host cells via the hemagglutinin (HA), a glycoprotein expressed on the viral envelope, and enters into the cytoplasm by receptor-mediated endocytosis. The viral genome is released and transported in to the nucleus, where transcription and replication take place. However, cellular factors affecting the influenza virus infection such as the cell cycle remain uncharacterized.
To resolve the influence of cell cycle on influenza virus infection, we performed a single-virus infection analysis using optical tweezers. Using this newly developed single-virus infection system, the fluorescence-labeled influenza virus was trapped on a microchip using a laser (1064 nm) at 0.6 W, transported, and released onto individual H292 human lung epithelial cells. Interestingly, the influenza virus attached selectively to cells in the G1-phase. To clarify the molecular differences between cells in G1- and S/G2/M-phase, we performed several physical and chemical assays. Results indicated that: 1) the membranes of cells in G1-phase contained greater amounts of sialic acids (glycoproteins) than the membranes of cells in S/G2/M-phase; 2) the membrane stiffness of cells in S/G2/M-phase is more rigid than those in G1-phase by measurement using optical tweezers; and 3) S/G2/M-phase cells contained higher content of Gb3, Gb4 and GlcCer than G1-phase cells by an assay for lipid composition.
A novel single-virus infection system was developed to characterize the difference in influenza virus susceptibility between G1- and S/G2/M-phase cells. Differences in virus binding specificity were associated with alterations in the lipid composition, sialic acid content, and membrane stiffness. This single-virus infection system will be useful for studying the infection mechanisms of other viruses.
DOI
Influenza virus attaches to sialic acid residues on the surface of host cells via the hemagglutinin (HA), a glycoprotein expressed on the viral envelope, and enters into the cytoplasm by receptor-mediated endocytosis. The viral genome is released and transported in to the nucleus, where transcription and replication take place. However, cellular factors affecting the influenza virus infection such as the cell cycle remain uncharacterized.
To resolve the influence of cell cycle on influenza virus infection, we performed a single-virus infection analysis using optical tweezers. Using this newly developed single-virus infection system, the fluorescence-labeled influenza virus was trapped on a microchip using a laser (1064 nm) at 0.6 W, transported, and released onto individual H292 human lung epithelial cells. Interestingly, the influenza virus attached selectively to cells in the G1-phase. To clarify the molecular differences between cells in G1- and S/G2/M-phase, we performed several physical and chemical assays. Results indicated that: 1) the membranes of cells in G1-phase contained greater amounts of sialic acids (glycoproteins) than the membranes of cells in S/G2/M-phase; 2) the membrane stiffness of cells in S/G2/M-phase is more rigid than those in G1-phase by measurement using optical tweezers; and 3) S/G2/M-phase cells contained higher content of Gb3, Gb4 and GlcCer than G1-phase cells by an assay for lipid composition.
A novel single-virus infection system was developed to characterize the difference in influenza virus susceptibility between G1- and S/G2/M-phase cells. Differences in virus binding specificity were associated with alterations in the lipid composition, sialic acid content, and membrane stiffness. This single-virus infection system will be useful for studying the infection mechanisms of other viruses.
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Attractive Optical Forces from Blackbody Radiation
M. Sonnleitner, M. Ritsch-Marte, and H. RitschBlackbody radiation around hot objects induces ac Stark shifts of the energy levels of nearby atoms and molecules. These shifts are roughly proportional to the fourth power of the temperature and induce a force decaying with the third power of the distance from the object. We explicitly calculate the resulting attractive blackbody optical dipole force for ground state hydrogen atoms. Surprisingly, this force can surpass the repulsive radiation pressure and actually pull the atoms against the radiation energy flow towards the surface with a force stronger than gravity. We exemplify the dominance of the “blackbody force” over gravity for hydrogen in a cloud of hot dust particles. This overlooked force appears relevant in various astrophysical scenarios, in particular, since analogous results hold for a wide class of other broadband radiation sources
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Optical formation and manipulation of particle and cell patterns using a tapered optical fiber
Hongbao Xin, Rui Xu, Baojun Li
A method for optical formation and controllable manipulation of particle and cell patterns using a tapered optical fiber is demonstrated. With a laser beam at 980-nm wavelength launched into the fiber, different sized silica particles were formed into particle patterns (both one-dimensional chains and two-dimensional arrays) with different particle numbers by optical binding. The formed particle patterns can be controllably manipulated in three dimensions. Using yeast cells as an example, it was demonstrated that the method is applicable for the formation of biological cell patterns, without damage to the yeast cell viability. This method provides a new facile way for biophotonic and biological researches with particles and cells in a highly organized manner.
DOI
A method for optical formation and controllable manipulation of particle and cell patterns using a tapered optical fiber is demonstrated. With a laser beam at 980-nm wavelength launched into the fiber, different sized silica particles were formed into particle patterns (both one-dimensional chains and two-dimensional arrays) with different particle numbers by optical binding. The formed particle patterns can be controllably manipulated in three dimensions. Using yeast cells as an example, it was demonstrated that the method is applicable for the formation of biological cell patterns, without damage to the yeast cell viability. This method provides a new facile way for biophotonic and biological researches with particles and cells in a highly organized manner.
DOI
H-Ras transfers from B to T cells via tunneling nanotubes
N Rainy, D Chetrit, V Rouger, H Vernitsky, O Rechavi, D Marguet, I Goldstein, M Ehrlich and Y Kloog
Lymphocytes form cell–cell connections by various mechanisms, including intercellular networks through actin-supported long-range plasma membrane (PM) extensions, termed tunneling nanotubes (TNTs). In this study, we tested in vitro whether TNTs form between human antigen-presenting B cells and T cells following cell contact and whether they enable the transfer of PM-associated proteins, such as green fluorescent protein (GFP)-tagged H-Ras (GFP-H-Ras). To address this question, we employed advanced techniques, including cell trapping by optical tweezers and live-cell imaging by 4D spinning-disk confocal microscopy. First, we showed that TNTs can form after optically trapped conjugated B and T cells are being pulled apart. Next, we determined by measuring fluorescence recovery after photobleaching that GFP-H-Ras diffuses freely in the membrane of TNTs that form spontaneously between B and T cells during coculturing. Importantly, by 4D time-lapse imaging, we showed that GFP-H-Ras-enriched PM patches accumulate at the junction between TNTs and the T-cell body and subsequently transfer to the T-cell surface. Furthermore, the PM patches adopted by T cells were enriched for another B-cell-derived transmembrane receptor, CD86. As predicted, the capacity of GFP-H-Ras to transfer between B and T cells, during coculturing, was dependent on its normal post-transcriptional lipidation and consequent PM anchorage. In summary, our data indicate that TNTs connecting B and T cells provide a hitherto undescribed route for the transfer of PM patches containing, for example, H-Ras from B to T cells.
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Self-Induced Torque in Hyperbolic Metamaterials
Pavel Ginzburg, Alexey V. Krasavin, Alexander N. Poddubny, Pavel A. Belov, Yuri S. Kivshar, and Anatoly V. Zayats
Optical forces constitute a fundamental phenomenon important in various fields of science, from astronomy to biology. Generally, intense external radiation sources are required to achieve measurable effects suitable for applications. Here we demonstrate that quantum emitters placed in a homogeneous anisotropic medium induce self-torques, aligning themselves in the well-defined direction determined by an anisotropy, in order to maximize their radiation efficiency. We develop a universal quantum-mechanical theory of self-induced torques acting on an emitter placed in a material environment. The theoretical framework is based on the radiation reaction approach utilizing the rigorous Langevin local quantization of electromagnetic excitations. We show more than 2 orders of magnitude enhancement of the self-torque by an anisotropic metamaterial with hyperbolic dispersion, having negative ratio of permittivity tensor components, in comparison with conventional anisotropic crystals with the highest naturally available anisotropy.
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Optical forces constitute a fundamental phenomenon important in various fields of science, from astronomy to biology. Generally, intense external radiation sources are required to achieve measurable effects suitable for applications. Here we demonstrate that quantum emitters placed in a homogeneous anisotropic medium induce self-torques, aligning themselves in the well-defined direction determined by an anisotropy, in order to maximize their radiation efficiency. We develop a universal quantum-mechanical theory of self-induced torques acting on an emitter placed in a material environment. The theoretical framework is based on the radiation reaction approach utilizing the rigorous Langevin local quantization of electromagnetic excitations. We show more than 2 orders of magnitude enhancement of the self-torque by an anisotropic metamaterial with hyperbolic dispersion, having negative ratio of permittivity tensor components, in comparison with conventional anisotropic crystals with the highest naturally available anisotropy.
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Spin Controlled Optical Radiation Pressure
Georgiy Tkachenko and Etienne Brasselet
We report on the full control of the optical radiation pressure at fixed photon flux and incident angle by the photon spin. This is done by using transparent chiral liquid crystal droplets that enable a strong coupling between the linear and angular degrees of freedom of a light field. From these results, we anticipate optical sorting of particles with different chirality as well as novel optical trapping and micromanipulation strategies.
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We report on the full control of the optical radiation pressure at fixed photon flux and incident angle by the photon spin. This is done by using transparent chiral liquid crystal droplets that enable a strong coupling between the linear and angular degrees of freedom of a light field. From these results, we anticipate optical sorting of particles with different chirality as well as novel optical trapping and micromanipulation strategies.
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Optofluidic manipulation of Escherichia coli in a microfluidic channel using an abruptly tapered optical fiber
Hongbao Xin, Yayi Li, Lingshan Li, Rui Xu, and Baojun Li
We report stable optical trapping and controlled manipulation of Escherichia coli cells in a microfluidic channel using an abruptly tapered optical fiber with 980-nm wavelength laser light launched. Stability of the trapping at different optical powers (10–70 mW) was demonstrated in fluids under different flow directions and velocities. The experimental results were supported by finite-element simulations and analytic calculations.
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We report stable optical trapping and controlled manipulation of Escherichia coli cells in a microfluidic channel using an abruptly tapered optical fiber with 980-nm wavelength laser light launched. Stability of the trapping at different optical powers (10–70 mW) was demonstrated in fluids under different flow directions and velocities. The experimental results were supported by finite-element simulations and analytic calculations.
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Monday, July 22, 2013
DNA unwinding heterogeneity by RecBCD results from static molecules able to equilibrate
Bian Liu, Ronald J. Baskin & Stephen C. Kowalczykowski
Single-molecule studies can overcome the complications of asynchrony and ensemble-averaging in bulk-phase measurements, provide mechanistic insights into molecular activities, and reveal interesting variations between individual molecules1, 2, 3. The application of these techniques to the RecBCD helicase of Escherichia coli has resolved some long-standing discrepancies, and has provided otherwise unattainable mechanistic insights into its enzymatic behaviour4, 5, 6. Enigmatically, the DNA unwinding rates of individual enzyme molecules are seen to vary considerably6, 7, 8, but the origin of this heterogeneity remains unknown. Here we investigate the physical basis for this behaviour. Although any individual RecBCD molecule unwound DNA at a constant rate for an average of approximately 30,000 steps, we discover that transiently halting a single enzyme–DNA complex by depleting Mg2+-ATP could change the subsequent rates of DNA unwinding by that enzyme after reintroduction to ligand. The proportion of molecules that changed rate increased exponentially with the duration of the interruption, with a half-life of approximately 1 second, suggesting that a conformational change occurred during the time that the molecule was arrested. The velocity after pausing an individual molecule was any velocity found in the starting distribution of the ensemble. We suggest that substrate binding stabilizes the enzyme in one of many equilibrium conformational sub-states that determine the rate-limiting translocation behaviour of each RecBCD molecule. Each stabilized sub-state can persist for the duration (approximately 1 minute) of processive unwinding of a DNA molecule, comprising tens of thousands of catalytic steps, each of which is much faster than the time needed for the conformational change required to alter kinetic behaviour. This ligand-dependent stabilization of rate-defining conformational sub-states results in seemingly static molecule-to-molecule variation in RecBCD helicase activity, but in fact reflects one microstate from the equilibrium ensemble that a single molecule manifests during an individual processive translocation event.
DOI
Single-molecule studies can overcome the complications of asynchrony and ensemble-averaging in bulk-phase measurements, provide mechanistic insights into molecular activities, and reveal interesting variations between individual molecules1, 2, 3. The application of these techniques to the RecBCD helicase of Escherichia coli has resolved some long-standing discrepancies, and has provided otherwise unattainable mechanistic insights into its enzymatic behaviour4, 5, 6. Enigmatically, the DNA unwinding rates of individual enzyme molecules are seen to vary considerably6, 7, 8, but the origin of this heterogeneity remains unknown. Here we investigate the physical basis for this behaviour. Although any individual RecBCD molecule unwound DNA at a constant rate for an average of approximately 30,000 steps, we discover that transiently halting a single enzyme–DNA complex by depleting Mg2+-ATP could change the subsequent rates of DNA unwinding by that enzyme after reintroduction to ligand. The proportion of molecules that changed rate increased exponentially with the duration of the interruption, with a half-life of approximately 1 second, suggesting that a conformational change occurred during the time that the molecule was arrested. The velocity after pausing an individual molecule was any velocity found in the starting distribution of the ensemble. We suggest that substrate binding stabilizes the enzyme in one of many equilibrium conformational sub-states that determine the rate-limiting translocation behaviour of each RecBCD molecule. Each stabilized sub-state can persist for the duration (approximately 1 minute) of processive unwinding of a DNA molecule, comprising tens of thousands of catalytic steps, each of which is much faster than the time needed for the conformational change required to alter kinetic behaviour. This ligand-dependent stabilization of rate-defining conformational sub-states results in seemingly static molecule-to-molecule variation in RecBCD helicase activity, but in fact reflects one microstate from the equilibrium ensemble that a single molecule manifests during an individual processive translocation event.
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Near-field electromagnetic trapping through curl-spin forces
Iñigo Liberal, Iñigo Ederra, Ramón Gonzalo, and Richard W. Ziolkowski
Near-field electromagnetic trapping of particles is generally obtained by means of gradient forces. In this paper, we discuss the attractive behavior of curl-spin forces, as well as their potential for near-field electromagnetic trapping and manipulation. It is demonstrated that curl-spin forces enable the trapping of particles operating at their resonant frequency. Such phenomena can be exploited to design more efficient and selective electromagnetic traps, to boost near-field energy exchange systems, and to bring stability to coupled resonant radiators. It also is illustrated how the balance between the gradient, radiation pressure, and curl-spin force components leads to the formation of zero-force rings around their sources, which explicitly demarcate the trapping regions. Analytical and numerical analyses are presented to assess the stability of the trapping mechanism.
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Near-field electromagnetic trapping of particles is generally obtained by means of gradient forces. In this paper, we discuss the attractive behavior of curl-spin forces, as well as their potential for near-field electromagnetic trapping and manipulation. It is demonstrated that curl-spin forces enable the trapping of particles operating at their resonant frequency. Such phenomena can be exploited to design more efficient and selective electromagnetic traps, to boost near-field energy exchange systems, and to bring stability to coupled resonant radiators. It also is illustrated how the balance between the gradient, radiation pressure, and curl-spin force components leads to the formation of zero-force rings around their sources, which explicitly demarcate the trapping regions. Analytical and numerical analyses are presented to assess the stability of the trapping mechanism.
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A computational tool to characterize particle tracking measurements in optical tweezers
Michael A Taylor and Warwick P Bowen
Here, we present a computational tool for optical tweezers which calculates the particle tracking signal measured with a quadrant detector and the shot-noise limit to position resolution. The tool is a piece of Matlab code which functions within the freely available Optical Tweezers Toolbox. It allows the measurements performed in most optical tweezer experiments to be theoretically characterized in a fast and easy manner. The code supports particles with arbitrary size, any optical fields and any combination of objective and condenser, and performs a full vector calculation of the relevant fields. Example calculations are presented which show the tracking signals for different particles, and the shot-noise limit to position sensitivity as a function of the effective condenser NA.
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Here, we present a computational tool for optical tweezers which calculates the particle tracking signal measured with a quadrant detector and the shot-noise limit to position resolution. The tool is a piece of Matlab code which functions within the freely available Optical Tweezers Toolbox. It allows the measurements performed in most optical tweezer experiments to be theoretically characterized in a fast and easy manner. The code supports particles with arbitrary size, any optical fields and any combination of objective and condenser, and performs a full vector calculation of the relevant fields. Example calculations are presented which show the tracking signals for different particles, and the shot-noise limit to position sensitivity as a function of the effective condenser NA.
DOI
Saturday, July 20, 2013
Resonance optical manipulation of nano-objects based on nonlinear optical response
Tetsuhiro Kudo and Hajime Ishihara
Optical manipulation is a technique to control the mechanical motion of small objects by using electromagnetic radiation force. Optical tweezers are the most popular tool to trap and move microparticles suspended in a medium. Recent interest has been shifting to manipulating nano-objects considerably smaller than the wavelength of light. Since the radiation force exerted on nano-objects is extremely small, an innovative method is necessary to make this concept feasible. Utilizing the resonant optical response of the objects to electronic transitions is one of the promising ways to approach nanoscale optical manipulation, and several advances in this direction have been made recently. Despite experimental studies on resonance optical tweezers showing favorable results, conventional theories have been unable to explain the results though demonstrations of resonant manipulations for traveling and standing waves have shown favorable results. In the present article, we provide a perspective view of resonance optical manipulation based on nonlinear optical response that we have recently proposed. This idea coherently elucidates recently reported puzzling phenomena appearing in studies concerning resonance optical tweezers that contradict the conventional understanding of resonance optical trapping. Further, this concept opens up the possibility to develop potentially powerful manipulation techniques because the nonlinear optical response involves processes with considerably greater degrees of freedom than those of linear optical response. As examples, we propose a method for trapping single organic molecules that is more effective than ever before, selectively pulling the molecules with a particular transition energy, and our proposed method allows for high-spatial-resolution optical manipulation beyond the diffraction limit.
DOI
Optical manipulation is a technique to control the mechanical motion of small objects by using electromagnetic radiation force. Optical tweezers are the most popular tool to trap and move microparticles suspended in a medium. Recent interest has been shifting to manipulating nano-objects considerably smaller than the wavelength of light. Since the radiation force exerted on nano-objects is extremely small, an innovative method is necessary to make this concept feasible. Utilizing the resonant optical response of the objects to electronic transitions is one of the promising ways to approach nanoscale optical manipulation, and several advances in this direction have been made recently. Despite experimental studies on resonance optical tweezers showing favorable results, conventional theories have been unable to explain the results though demonstrations of resonant manipulations for traveling and standing waves have shown favorable results. In the present article, we provide a perspective view of resonance optical manipulation based on nonlinear optical response that we have recently proposed. This idea coherently elucidates recently reported puzzling phenomena appearing in studies concerning resonance optical tweezers that contradict the conventional understanding of resonance optical trapping. Further, this concept opens up the possibility to develop potentially powerful manipulation techniques because the nonlinear optical response involves processes with considerably greater degrees of freedom than those of linear optical response. As examples, we propose a method for trapping single organic molecules that is more effective than ever before, selectively pulling the molecules with a particular transition energy, and our proposed method allows for high-spatial-resolution optical manipulation beyond the diffraction limit.
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Optical stretching as a tool to investigate the mechanical properties of lipid bilayers
Mehmet E. Solmaz, Shalene Sankhagowit, Roshni Biswas, Camilo A. Mejia, Michelle L. Povinelli and Noah Malmstadt
Measurements of lipid bilayer bending modulus by various techniques produce widely divergent results. We attempt to resolve some of this ambiguity by measuring bending modulus in a system that can rapidly process large numbers of samples, yielding population statistics. This system is based on optical stretching of giant unilamellar vesicles (GUVs) in a microfluidic dual-beam optical trap (DBOT). The microfluidic DBOT system is used here to measure three populations of GUVs with distinct lipid compositions. We find that gel-phase membranes are significantly stiffer than liquid-phase membranes, consistent with previous reports. We also find that the addition of cholesterol does not alter the bending modulus of membranes composed of a monounsaturated phospholipid.
DOI
Measurements of lipid bilayer bending modulus by various techniques produce widely divergent results. We attempt to resolve some of this ambiguity by measuring bending modulus in a system that can rapidly process large numbers of samples, yielding population statistics. This system is based on optical stretching of giant unilamellar vesicles (GUVs) in a microfluidic dual-beam optical trap (DBOT). The microfluidic DBOT system is used here to measure three populations of GUVs with distinct lipid compositions. We find that gel-phase membranes are significantly stiffer than liquid-phase membranes, consistent with previous reports. We also find that the addition of cholesterol does not alter the bending modulus of membranes composed of a monounsaturated phospholipid.
DOI
Friday, July 19, 2013
Pattern matching based active optical sorting of colloids/cells
R S Verma, R Dasgupta, S Ahlawat, N Kumar, A Uppal and P K Gupta
We report active optical sorting of colloids/cells by employing a cross correlation based pattern matching technique for selection of the desired objects and thereafter sorting using dynamically controllable holographic optical traps. The problem of possible collision between the different sets of objects during sorting was avoided by raising one set of particles to a different plane. We also present the results obtained on using this approach for some representative applications such as sorting of silica particles of two different sizes, of closely packed colloids and of white blood cells and red blood cells from a mixture of the two.
DOI
We report active optical sorting of colloids/cells by employing a cross correlation based pattern matching technique for selection of the desired objects and thereafter sorting using dynamically controllable holographic optical traps. The problem of possible collision between the different sets of objects during sorting was avoided by raising one set of particles to a different plane. We also present the results obtained on using this approach for some representative applications such as sorting of silica particles of two different sizes, of closely packed colloids and of white blood cells and red blood cells from a mixture of the two.
DOI
Laser-induced fusion of human embryonic stem cells with optical tweezers
Shuxun Chen, Jinping Cheng, Chi-Wing Kong, Xiaolin Wang, Shuk Han Cheng, Ronald A. Li, and Dong Sun
We report a study on the laser-induced fusion of human embryonic stem cells (hESCs) at the single-cell level. Cells were manipulated by optical tweezers and fused under irradiation with pulsed UV laser at 355 nm. Successful fusion was indicated by green fluorescence protein transfer. The influence of laser pulse energy on the fusion efficiency was investigated. The fused products were viable as gauged by live cell staining. Successful fusion of hESCs with somatic cells was also demonstrated. The reported fusion outcome may facilitate studies of cell differentiation, maturation, and reprogramming.
DOI
We report a study on the laser-induced fusion of human embryonic stem cells (hESCs) at the single-cell level. Cells were manipulated by optical tweezers and fused under irradiation with pulsed UV laser at 355 nm. Successful fusion was indicated by green fluorescence protein transfer. The influence of laser pulse energy on the fusion efficiency was investigated. The fused products were viable as gauged by live cell staining. Successful fusion of hESCs with somatic cells was also demonstrated. The reported fusion outcome may facilitate studies of cell differentiation, maturation, and reprogramming.
DOI
Application of femtosecond laser pulses in biomedical cell technologies
I. V. Ilina, A. V. Ovchinnikov, D. S. Sitnikov, M. M. Rakityanskiy, M. B. Agranat, Y. V. Khramova, M. L. Semenova
The results are presented of the works in the field of development of equipment, investigation techniques, and technologies for biology and medicine performed in the Joint Institute for High Temperatures of the Russian Academy of Scienses (JIHT RAS). On the base of the new generation infrared femtosecond lasers, the experimental models are developed and manufactured of laser tweezers, scalpel, and the “tweezers-scalpel” combined system. The results are presented of the experimental studies on the noncontact mammalian cell fusion (blastomeres of mouse embryos on day 1.5 of development) by means of the femtosecond laser pulses.
DOI
The results are presented of the works in the field of development of equipment, investigation techniques, and technologies for biology and medicine performed in the Joint Institute for High Temperatures of the Russian Academy of Scienses (JIHT RAS). On the base of the new generation infrared femtosecond lasers, the experimental models are developed and manufactured of laser tweezers, scalpel, and the “tweezers-scalpel” combined system. The results are presented of the experimental studies on the noncontact mammalian cell fusion (blastomeres of mouse embryos on day 1.5 of development) by means of the femtosecond laser pulses.
DOI
Thursday, July 18, 2013
Reshaping of the conformational search of a protein by the chaperone trigger factor
Alireza Mashaghi, Günter Kramer, Philipp Bechtluft, Beate Zachmann-Brand, Arnold J. M. Driessen, Bernd Bukau & Sander J. Tans
Protein folding is often described as a search process, in which polypeptides explore different conformations to find their native structure. Molecular chaperones are known to improve folding yields by suppressing aggregation between polypeptides before this conformational search starts1, 2, as well as by rescuing misfolds after it ends1, 3. Although chaperones have long been speculated to also affect the conformational search itself—by reshaping the underlying folding landscape along the folding trajectory4, 5—direct experimental evidence has been scarce so far. In Escherichia coli, the general chaperone trigger factor6, 7, 8 (TF) could play such a role. TF has been shown to interact with nascent chains at the ribosome9, 10, with polypeptides released from the ribosome into the cytosol11, and with fully folded proteins before their assembly into larger complexes12. To investigate the effect of TF from E. coli on the conformational search of polypeptides to their native state, we investigated individual maltose binding protein (MBP) molecules using optical tweezers. Here we show that TF binds folded structures smaller than one domain, which are then stable for seconds and ultimately convert to the native state. Moreover, TF stimulates native folding in constructs of repeated MBP domains. The results indicate that TF promotes correct folding by protecting partially folded states from distant interactions that produce stable misfolded states. As TF interacts with most newly synthesized proteins in E. coli, we expect these findings to be of general importance in understanding protein folding pathways.
DOI
Protein folding is often described as a search process, in which polypeptides explore different conformations to find their native structure. Molecular chaperones are known to improve folding yields by suppressing aggregation between polypeptides before this conformational search starts1, 2, as well as by rescuing misfolds after it ends1, 3. Although chaperones have long been speculated to also affect the conformational search itself—by reshaping the underlying folding landscape along the folding trajectory4, 5—direct experimental evidence has been scarce so far. In Escherichia coli, the general chaperone trigger factor6, 7, 8 (TF) could play such a role. TF has been shown to interact with nascent chains at the ribosome9, 10, with polypeptides released from the ribosome into the cytosol11, and with fully folded proteins before their assembly into larger complexes12. To investigate the effect of TF from E. coli on the conformational search of polypeptides to their native state, we investigated individual maltose binding protein (MBP) molecules using optical tweezers. Here we show that TF binds folded structures smaller than one domain, which are then stable for seconds and ultimately convert to the native state. Moreover, TF stimulates native folding in constructs of repeated MBP domains. The results indicate that TF promotes correct folding by protecting partially folded states from distant interactions that produce stable misfolded states. As TF interacts with most newly synthesized proteins in E. coli, we expect these findings to be of general importance in understanding protein folding pathways.
DOI
Manipulation and assembly of small objects in liquid crystals by dynamical disorganizing effect of push-pull-azobenzene-dye
Seiji Kurihara, Kazuhiro Ohta, Takahiro Oda, Ryo Izumi, Yutaka Kuwahara, Tomonari Ogata, and Sun-Nam Kim
The phase transition of a nematic liquid crystal containing a push-pull azobenzene dye could be induced efficiently during irradiation with visible light. The dynamical disorganizing effect of the push-pull azobenzene dye on the liquid crystalline order through its trans-cis-trans photoisomerizaion cycle under visible light was contributed to the efficient phase transition. Then, the effects of light irradiation on the motion of small objects dispersed in the liquid crystals containing the push-pull azobenzene were explored, and the manipulation and assembly of those objects were successfully achieved in the nematic phase but also in the smectic phase. The combination of the photo-controlled dynamical change in the liquid crystalline order and the intrinsic self-assembly property of a liquid crystal is promising for use in technologies that require not only the organization of small objects but also the photo-driving of nano- and micro-sized mechanical materials.
DOI
The phase transition of a nematic liquid crystal containing a push-pull azobenzene dye could be induced efficiently during irradiation with visible light. The dynamical disorganizing effect of the push-pull azobenzene dye on the liquid crystalline order through its trans-cis-trans photoisomerizaion cycle under visible light was contributed to the efficient phase transition. Then, the effects of light irradiation on the motion of small objects dispersed in the liquid crystals containing the push-pull azobenzene were explored, and the manipulation and assembly of those objects were successfully achieved in the nematic phase but also in the smectic phase. The combination of the photo-controlled dynamical change in the liquid crystalline order and the intrinsic self-assembly property of a liquid crystal is promising for use in technologies that require not only the organization of small objects but also the photo-driving of nano- and micro-sized mechanical materials.
DOI
Laser Trapping and Crystallization Dynamics of l-Phenylalanine at Solution Surface
Ken-ichi Yuyama, Teruki Sugiyama, and Hiroshi Masuhara
We present laser trapping behavior of l-phenylalanine (l-Phe) at a surface of its unsaturated aqueous solution by a focused continuous-wave (CW) near-infrared (NIR) laser beam. Upon the irradiation into the solution surface, laser trapping of the liquid-like clusters is induced concurrently with local laser heating, forming an anhydrous plate-like crystal at the focal spot. The following laser irradiation into a central part of the plate-like crystal leads to laser trapping at the crystal surface not only for l-Phe molecules/clusters but also for polystyrene (PS) particles. The particles are closely packed at crystal edges despite that the crystal surface is not illuminated by the laser directly. The molecules/clusters are also gathered and adsorbed to the crystal surface, leading to crystal growth. The trapping dynamics and mechanism are discussed in view of optical potential formed at the crystal surface by light propagation inside the crystal.
DOI
We present laser trapping behavior of l-phenylalanine (l-Phe) at a surface of its unsaturated aqueous solution by a focused continuous-wave (CW) near-infrared (NIR) laser beam. Upon the irradiation into the solution surface, laser trapping of the liquid-like clusters is induced concurrently with local laser heating, forming an anhydrous plate-like crystal at the focal spot. The following laser irradiation into a central part of the plate-like crystal leads to laser trapping at the crystal surface not only for l-Phe molecules/clusters but also for polystyrene (PS) particles. The particles are closely packed at crystal edges despite that the crystal surface is not illuminated by the laser directly. The molecules/clusters are also gathered and adsorbed to the crystal surface, leading to crystal growth. The trapping dynamics and mechanism are discussed in view of optical potential formed at the crystal surface by light propagation inside the crystal.
DOI
Sunday, July 14, 2013
Compensation of the optically induced Lorentz force in a homogeneous optical medium
Vladimir Torchigin, Alexander Torchigin
It is shown on the basis of different approaches that the density of optically induced forces applied to a homogeneous optical medium embedded in a simplest 1D structure in a form of a plane optical resonator is equal to zero. In particular, the density forces calculated on the base of the energetic approach, where no assumptions about physical nature of optically induced forces are used, are also equal to zero. At the same time the same forces calculated by means of the approach based on the Lorentz force are different from zero. A conclusion is derived that there is an additional type of optically induced force which compensates the Lorentz density forces. Thus, the Lorentz force approach used for calculation of the density of optically induced force is inconsistent.
DOI
It is shown on the basis of different approaches that the density of optically induced forces applied to a homogeneous optical medium embedded in a simplest 1D structure in a form of a plane optical resonator is equal to zero. In particular, the density forces calculated on the base of the energetic approach, where no assumptions about physical nature of optically induced forces are used, are also equal to zero. At the same time the same forces calculated by means of the approach based on the Lorentz force are different from zero. A conclusion is derived that there is an additional type of optically induced force which compensates the Lorentz density forces. Thus, the Lorentz force approach used for calculation of the density of optically induced force is inconsistent.
DOI
Investigation on specific solutions of Gerchberg–Saxton algorithm
Pasquale Memmolo
, Lisa Miccio, Francesco Merola, Antonio Paciello, Valerio Embrione, Sabato Fusco, Pietro Ferraro, Paolo Antonio Netti
The most popular method used to generate the Computer Generated Holograms (CGH) is the Gerchberg–Saxton (GS) algorithm. GS computes an approximation of the desired beam shape, and consequently, some distortions may arise. Although many algorithms have been proposed, exact methods to overcome the problem completely do not yet exist. Here we show, for the first time to best of our knowledge, that in some specific configurations exact solutions of the GS algorithm can be achieved so as to produce a limited number of light intensity spots in a clean array. The basic concept is described and both numerical as well as experimental implementations are provided.
DOI
, Lisa Miccio, Francesco Merola, Antonio Paciello, Valerio Embrione, Sabato Fusco, Pietro Ferraro, Paolo Antonio NettiThe most popular method used to generate the Computer Generated Holograms (CGH) is the Gerchberg–Saxton (GS) algorithm. GS computes an approximation of the desired beam shape, and consequently, some distortions may arise. Although many algorithms have been proposed, exact methods to overcome the problem completely do not yet exist. Here we show, for the first time to best of our knowledge, that in some specific configurations exact solutions of the GS algorithm can be achieved so as to produce a limited number of light intensity spots in a clean array. The basic concept is described and both numerical as well as experimental implementations are provided.
DOI
Saturday, July 13, 2013
Mode division multiplexing technology for single-fiber optical trapping axial-position adjustment
Zhihai Liu, Lei Wang, Peibo Liang, Yu Zhang, Jun Yang, and Libo Yuan
We demonstrate trapped yeast cell axial-position adjustment without moving the optical fiber in a single-fiber optical trapping system. The dynamic axial-position adjustment is realized by controlling the power ratio of the fundamental mode beam (LP01) and the low-order mode beam (LP11) generated in a normal single-core fiber. In order to separate the trapping positions produced by the two mode beams, we fabricate a special fiber tapered tip with a selective two-step method. A yeast cell of 6 μm diameter is moved along the optical axis direction for a distance of ∼3 μm. To the best of our knowledge, this is the first demonstration of the trapping position adjustment without moving the fiber for single-fiber optical tweezers. The excitation and utilization of multimode beams in a single fiber constitutes a new development for single-fiber optical trapping and makes possible more practical applications in biomedical research fields.
DOI
We demonstrate trapped yeast cell axial-position adjustment without moving the optical fiber in a single-fiber optical trapping system. The dynamic axial-position adjustment is realized by controlling the power ratio of the fundamental mode beam (LP01) and the low-order mode beam (LP11) generated in a normal single-core fiber. In order to separate the trapping positions produced by the two mode beams, we fabricate a special fiber tapered tip with a selective two-step method. A yeast cell of 6 μm diameter is moved along the optical axis direction for a distance of ∼3 μm. To the best of our knowledge, this is the first demonstration of the trapping position adjustment without moving the fiber for single-fiber optical tweezers. The excitation and utilization of multimode beams in a single fiber constitutes a new development for single-fiber optical trapping and makes possible more practical applications in biomedical research fields.
DOI
Thursday, July 11, 2013
Optical tweezers assisted quantitative phase imaging led to thickness mapping of red blood cells
Nelson Cardenas and Samarendra K. MohantyQuantitative phase microscopy (QPM) allows dynamic mapping of optical path length of microscopic samples with high temporal and axial resolution. However, decoupling of the geometric thickness from the refractive index in phase measurements is challenging. Here, we report use of optical tweezers combined with QPM for decoupling geometric thickness from the refractive index. This is demonstrated by orienting the microscopic sample (red blood cell) by optical tweezers and imaging the phase at various orientations. Since optical tweezers can orient wide variety of micro and nanoscopic objects, this integrated method can be employed to accurately determine their physical properties.
DOI
DOI
Tuesday, July 9, 2013
Membrane Tension in Rapidly Moving Cells Is Determined by Cytoskeletal Forces
Arnon D. Lieber, Shlomit Yehudai-Resheff, Erin L. Barnhart, Julie A. Theriot, Kinneret Keren
Membrane tension plays an essential role in cell motility. The load imposed by the tensed membrane restrains actin polymerization, promotes rear retraction, and influences membrane transport. Moreover, membrane tension is crucial for large-scale coordination of cell boundary dynamics. Despite its importance, little is known about how membrane tension is set and regulated in cells. The prevailing hypothesis is that membrane tension is largely controlled by membrane-cytoskeleton adhesion and/or changes in membrane area.In this work, we measure the apparent membrane tension in rapidly moving fish epithelial keratocytes under normal and perturbed conditions with a tether-pulling assay. We find that enlargement of the cell surface area by fusion with giant unilamellar vesicles (GUVs) has only minor effects on membrane tension and on cell movement. However, modulation of the cytoskeletal forces has a substantial influence on tension: reduction of the actin-pushing forces along the cell’s leading edge leads to a significant decrease in membrane tension, whereas increase of the strength of adhesion and/or decrease of myosin-induced contraction leads to higher tension.
We find that the membrane tension in rapidly moving keratocytes is primarily determined by a mechanical force balance between the cell membrane and cytoskeletal forces. Our results highlight the role of membrane tension as a global mechanical regulator of cell behavior.
DOI
Membrane tension plays an essential role in cell motility. The load imposed by the tensed membrane restrains actin polymerization, promotes rear retraction, and influences membrane transport. Moreover, membrane tension is crucial for large-scale coordination of cell boundary dynamics. Despite its importance, little is known about how membrane tension is set and regulated in cells. The prevailing hypothesis is that membrane tension is largely controlled by membrane-cytoskeleton adhesion and/or changes in membrane area.In this work, we measure the apparent membrane tension in rapidly moving fish epithelial keratocytes under normal and perturbed conditions with a tether-pulling assay. We find that enlargement of the cell surface area by fusion with giant unilamellar vesicles (GUVs) has only minor effects on membrane tension and on cell movement. However, modulation of the cytoskeletal forces has a substantial influence on tension: reduction of the actin-pushing forces along the cell’s leading edge leads to a significant decrease in membrane tension, whereas increase of the strength of adhesion and/or decrease of myosin-induced contraction leads to higher tension.
We find that the membrane tension in rapidly moving keratocytes is primarily determined by a mechanical force balance between the cell membrane and cytoskeletal forces. Our results highlight the role of membrane tension as a global mechanical regulator of cell behavior.
DOI
Membrane Elastic Properties and Cell Function
Bruno Pontes, Yareni Ayala, Anna Carolina C. Fonseca, Luciana F. Romão, Racκele F. Amaral, Leonardo T. Salgado, Flavia R. Lima, Marcos Farina, Nathan B. Viana, Vivaldo Moura-Neto, H. Moysés Nussenzveig
Recent studies indicate that the cell membrane, interacting with its attached cytoskeleton, is an important regulator of cell function, exerting and responding to forces. We investigate this relationship by looking for connections between cell membrane elastic properties, especially surface tension and bending modulus, and cell function. Those properties are measured by pulling tethers from the cell membrane with optical tweezers. Their values are determined for all major cell types of the central nervous system, as well as for macrophage. Astrocytes and glioblastoma cells, which are considerably more dynamic than neurons, have substantially larger surface tensions. Resting microglia, which continually scan their environment through motility and protrusions, have the highest elastic constants, with values similar to those for resting macrophage. For both microglia and macrophage, we find a sharp softening of bending modulus between their resting and activated forms, which is very advantageous for their acquisition of phagocytic functions upon activation. We also determine the elastic constants of pure cell membrane, with no attached cytoskeleton. For all cell types, the presence of F-actin within tethers, contrary to conventional wisdom, is confirmed. Our findings suggest the existence of a close connection between membrane elastic constants and cell function.
DOI
Recent studies indicate that the cell membrane, interacting with its attached cytoskeleton, is an important regulator of cell function, exerting and responding to forces. We investigate this relationship by looking for connections between cell membrane elastic properties, especially surface tension and bending modulus, and cell function. Those properties are measured by pulling tethers from the cell membrane with optical tweezers. Their values are determined for all major cell types of the central nervous system, as well as for macrophage. Astrocytes and glioblastoma cells, which are considerably more dynamic than neurons, have substantially larger surface tensions. Resting microglia, which continually scan their environment through motility and protrusions, have the highest elastic constants, with values similar to those for resting macrophage. For both microglia and macrophage, we find a sharp softening of bending modulus between their resting and activated forms, which is very advantageous for their acquisition of phagocytic functions upon activation. We also determine the elastic constants of pure cell membrane, with no attached cytoskeleton. For all cell types, the presence of F-actin within tethers, contrary to conventional wisdom, is confirmed. Our findings suggest the existence of a close connection between membrane elastic constants and cell function.
DOI
Monday, July 8, 2013
Plasma membrane tension orchestrates membrane trafficking, cytoskeletal remodeling, and biochemical signaling during phagocytosis
Thomas A. Masters, Bruno Pontes, Virgile Viasnoff, You Li, and Nils C. Gauthier
Phagocytes clear the body of undesirable particles such as infectious agents and debris. To extend pseudopods over the surface of targeted particles during engulfment, cells must change shape through extensive membrane and cytoskeleton remodeling. We observed that pseudopod extension occurred in two phases. In the first phase, pseudopods extended rapidly, with actin polymerization pushing the plasma membrane forward. The second phase occurred once the membrane area from preexisting reservoirs was depleted, leading to increased membrane tension. Increased tension directly altered the small Rho GTPase Rac1, 3′-phosphoinositide, and cytoskeletal organization. Furthermore, it activated exocytosis of vesicles containing GPI-anchored proteins, increasing membrane area and phagocytosis efficiency for large particles. We thus propose that, during phagocytosis, membrane remodeling, cytoskeletal organization, and biochemical signaling are orchestrated by the mechanical signal of membrane tension. These results put a simple mechanical signal at the heart of understanding immunological responses.
Phagocytes clear the body of undesirable particles such as infectious agents and debris. To extend pseudopods over the surface of targeted particles during engulfment, cells must change shape through extensive membrane and cytoskeleton remodeling. We observed that pseudopod extension occurred in two phases. In the first phase, pseudopods extended rapidly, with actin polymerization pushing the plasma membrane forward. The second phase occurred once the membrane area from preexisting reservoirs was depleted, leading to increased membrane tension. Increased tension directly altered the small Rho GTPase Rac1, 3′-phosphoinositide, and cytoskeletal organization. Furthermore, it activated exocytosis of vesicles containing GPI-anchored proteins, increasing membrane area and phagocytosis efficiency for large particles. We thus propose that, during phagocytosis, membrane remodeling, cytoskeletal organization, and biochemical signaling are orchestrated by the mechanical signal of membrane tension. These results put a simple mechanical signal at the heart of understanding immunological responses.
Dynamics of mechanosensing in the bacterial flagellar motor
Pushkar P. Lele, Basarab G. Hosu, and Howard C. Berg
Mechanosensing by flagella is thought to trigger bacterial swarmer-cell differentiation, an important step in pathogenesis. How flagellar motors sense mechanical stimuli is not known. To study this problem, we suddenly increased the viscous drag on motors by a large factor, from very low loads experienced by motors driving hooks or hooks with short filament stubs, to high loads, experienced by motors driving tethered cells or 1-μm latex beads. From the initial speed (after the load change), we inferred that motors running at very low loads are driven by one or at most two force-generating units. Following the load change, motors gradually adapted by increasing their speeds in a stepwise manner (over a period of a few minutes). Motors initially spun exclusively counterclockwise, but then increased the fraction of time that they spun clockwise over a time span similar to that observed for adaptation in speed. Single-motor total internal reflection fluorescence imaging of YFP–MotB (part of a stator force-generating unit) confirmed that the response to sudden increments in load occurred by the addition of new force-generating units. We estimate that 6–11 force-generating units drive motors at high loads. Wild-type motors and motors locked in the clockwise or counterclockwise state behaved in a similar manner, as did motors in cells deleted for the motor protein gene fliL or for genes in the chemotaxis signaling pathway. Thus, it appears that stators themselves act as dynamic mechanosensors. They change their structure in response to changes in external load. How such changes might impact cellular functions other than motility remains an interesting question.
Mechanosensing by flagella is thought to trigger bacterial swarmer-cell differentiation, an important step in pathogenesis. How flagellar motors sense mechanical stimuli is not known. To study this problem, we suddenly increased the viscous drag on motors by a large factor, from very low loads experienced by motors driving hooks or hooks with short filament stubs, to high loads, experienced by motors driving tethered cells or 1-μm latex beads. From the initial speed (after the load change), we inferred that motors running at very low loads are driven by one or at most two force-generating units. Following the load change, motors gradually adapted by increasing their speeds in a stepwise manner (over a period of a few minutes). Motors initially spun exclusively counterclockwise, but then increased the fraction of time that they spun clockwise over a time span similar to that observed for adaptation in speed. Single-motor total internal reflection fluorescence imaging of YFP–MotB (part of a stator force-generating unit) confirmed that the response to sudden increments in load occurred by the addition of new force-generating units. We estimate that 6–11 force-generating units drive motors at high loads. Wild-type motors and motors locked in the clockwise or counterclockwise state behaved in a similar manner, as did motors in cells deleted for the motor protein gene fliL or for genes in the chemotaxis signaling pathway. Thus, it appears that stators themselves act as dynamic mechanosensors. They change their structure in response to changes in external load. How such changes might impact cellular functions other than motility remains an interesting question.
Friday, July 5, 2013
Permanent Fixing or Reversible Trapping and Release of DNA Micropatterns on a Gold Nanostructure Using Continuous-Wave or Femtosecond-Pulsed Near-Infrared Laser Light
Tatsuya Shoji, Junki Saitoh, Noboru Kitamura, Fumika Nagasawa, Kei Murakoshi, Hiroaki Yamauchi, Syoji Ito, Hiroshi Miyasaka, Hajime Ishihara, and Yasuyuki Tsuboi
The use of localized surface plasmons (LSPs) for highly sensitive biosensors has already been investigated, and they are currently being applied for the optical manipulation of small nanoparticles. The objective of this work was the optical trapping of λ-DNA on a metallic nanostructure with femtosecond-pulsed (fs) laser irradiation. Continuous-wave laser irradiation, which is generally used for plasmon excitation, not only increased the electromagnetic field intensity but also generated heat around the nanostructure, causing the DNA to become permanently fixed on the plasmonic substrate. Using fs laser irradiation, on the other hand, the reversible trapping and release of the DNA was achieved by switching the fs laser irradiation on and off. This trap-and-release behavior was clearly observed using a fluorescence microscope. This technique can also be used to manipulate other biomolecules such as nucleic acids, proteins, and polysaccharides and will prove to be a useful tool in the fabrication of biosensors.
DOI
The use of localized surface plasmons (LSPs) for highly sensitive biosensors has already been investigated, and they are currently being applied for the optical manipulation of small nanoparticles. The objective of this work was the optical trapping of λ-DNA on a metallic nanostructure with femtosecond-pulsed (fs) laser irradiation. Continuous-wave laser irradiation, which is generally used for plasmon excitation, not only increased the electromagnetic field intensity but also generated heat around the nanostructure, causing the DNA to become permanently fixed on the plasmonic substrate. Using fs laser irradiation, on the other hand, the reversible trapping and release of the DNA was achieved by switching the fs laser irradiation on and off. This trap-and-release behavior was clearly observed using a fluorescence microscope. This technique can also be used to manipulate other biomolecules such as nucleic acids, proteins, and polysaccharides and will prove to be a useful tool in the fabrication of biosensors.
DOI
Thursday, July 4, 2013
Quantitative determination of optical trapping strength and viscoelastic moduli inside living cells
Josep Mas, Andrew C Richardson, S Nader S Reihani, Lene B Oddershede and Kirstine Berg-Sørensen
With the success of in vitro single-molecule force measurements obtained in recent years, the next step is to perform quantitative force measurements inside a living cell. Optical traps have proven excellent tools for manipulation, also in vivo, where they can be essentially non-invasive under correct wavelength and exposure conditions. It is a pre-requisite for in vivo quantitative force measurements that a precise and reliable force calibration of the tweezers is performed. There are well-established calibration protocols in purely viscous environments; however, as the cellular cytoplasm is viscoelastic, it would be incorrect to use a calibration procedure relying on a viscous environment. Here we demonstrate a method to perform a correct force calibration inside a living cell. This method (theoretically proposed in Fischer and Berg-Sørensen (2007 J. Opt. A: Pure Appl. Opt. 9 S239)) takes into account the viscoelastic properties of the cytoplasm and relies on a combination of active and passive recordings of the motion of the cytoplasmic object of interest. The calibration procedure allows us to extract absolute values for the viscoelastic moduli of the living cell cytoplasm as well as the force constant describing the optical trap, thus paving the way for quantitative force measurements inside the living cell. Here, we determine both the spring constant of the optical trap and the elastic contribution from the cytoplasm, influencing the motion of naturally occurring tracer particles. The viscoelastic moduli that we find are of the same order of magnitude as moduli found in other cell types by alternative methods.
DOI
With the success of in vitro single-molecule force measurements obtained in recent years, the next step is to perform quantitative force measurements inside a living cell. Optical traps have proven excellent tools for manipulation, also in vivo, where they can be essentially non-invasive under correct wavelength and exposure conditions. It is a pre-requisite for in vivo quantitative force measurements that a precise and reliable force calibration of the tweezers is performed. There are well-established calibration protocols in purely viscous environments; however, as the cellular cytoplasm is viscoelastic, it would be incorrect to use a calibration procedure relying on a viscous environment. Here we demonstrate a method to perform a correct force calibration inside a living cell. This method (theoretically proposed in Fischer and Berg-Sørensen (2007 J. Opt. A: Pure Appl. Opt. 9 S239)) takes into account the viscoelastic properties of the cytoplasm and relies on a combination of active and passive recordings of the motion of the cytoplasmic object of interest. The calibration procedure allows us to extract absolute values for the viscoelastic moduli of the living cell cytoplasm as well as the force constant describing the optical trap, thus paving the way for quantitative force measurements inside the living cell. Here, we determine both the spring constant of the optical trap and the elastic contribution from the cytoplasm, influencing the motion of naturally occurring tracer particles. The viscoelastic moduli that we find are of the same order of magnitude as moduli found in other cell types by alternative methods.
DOI
Adaptive optics in an optical trapping system for enhanced lateral trap stiffness at depth
M C Müllenbroich, N McAlinden and A J Wright
In optical trapping systems the trap stiffness, or spring constant, deteriorates dramatically with trap depth due to optical aberrations and system misalignment. This can severely hamper studies that employ optical tweezers to make accurate quantitative measurements. Here, a deformable membrane mirror is used, in conjunction with a random search algorithm, to correct for these aberrations by optimizing on a merit factor that is directly proportional to the trap stiffness. Previous studies have sought to address this issue but none have used a merit factor that is directly proportional to the trap stiffness. We demonstrate that the lateral trap stiffness, measured with and without aberration correction at increasing depths, improves throughout the trapping range of a conventional trap and allows us to extend the maximum depth at which we can trap from 136 to 166 μm. At a depth of 131 μm, trap stiffness improved by factors of 4.37 and 3.31 for the x- and y-axes respectively. The aberration correction resulted in deformable membrane mirror shapes where a single shape could be applied throughout a wide range of trap depths, showing significant improvement, and had the added benefit of making the lateral trapping forces more uniform in x and y.
DOI
In optical trapping systems the trap stiffness, or spring constant, deteriorates dramatically with trap depth due to optical aberrations and system misalignment. This can severely hamper studies that employ optical tweezers to make accurate quantitative measurements. Here, a deformable membrane mirror is used, in conjunction with a random search algorithm, to correct for these aberrations by optimizing on a merit factor that is directly proportional to the trap stiffness. Previous studies have sought to address this issue but none have used a merit factor that is directly proportional to the trap stiffness. We demonstrate that the lateral trap stiffness, measured with and without aberration correction at increasing depths, improves throughout the trapping range of a conventional trap and allows us to extend the maximum depth at which we can trap from 136 to 166 μm. At a depth of 131 μm, trap stiffness improved by factors of 4.37 and 3.31 for the x- and y-axes respectively. The aberration correction resulted in deformable membrane mirror shapes where a single shape could be applied throughout a wide range of trap depths, showing significant improvement, and had the added benefit of making the lateral trapping forces more uniform in x and y.
DOI
Tuesday, July 2, 2013
Analysis of diffuse K+ and Mg2+ ion binding to a two-base-pair kissing complex by single-molecule mechanical unfolding
Pan TX Li
The folding and stability of RNA tertiary interactions depends critically on cationic conditions. It is usually difficult, however, to isolate such effects on tertiary interactions from those on the entire RNA. By manipulating conformations of single RNA molecules using optical tweezers, we distinguished individual steps of breaking and forming of a two-base-pair kissing interaction from those of secondary folding. The binding of metal ions to the small tertiary structure appeared to be saturable with an apparent Kd of 160 mM for K+ and 1.5 mM for Mg2+. The kissing formation was estimated to be associated with binding of ~2-3 diffuse K+ or Mg2+ ions. At their saturated binding, Mg2+ provided ~3 kcal/mol more stabilizing energy to the structure than K+. Furthermore, the cations change the unkissing forces significantly more than the kissing ones. For example, the presence of Mg2+ ions increased the average unkissing force from 21 pN to 44 pN, surprisingly high for breaking merely two base pairs; in contrast, the mean kissing force was changed by only 4.5 pN. Interestingly, the differential salt effects on the transition forces were not caused by different changes in the height of the kinetic barriers, but were instead attributed to how different molecular structures respond to the applied force. Our results showed the importance of diffuse cation binding to the stability of tertiary interaction and demonstrated the utility of mechanical unfolding in studying tertiary interactions.
DOI
The folding and stability of RNA tertiary interactions depends critically on cationic conditions. It is usually difficult, however, to isolate such effects on tertiary interactions from those on the entire RNA. By manipulating conformations of single RNA molecules using optical tweezers, we distinguished individual steps of breaking and forming of a two-base-pair kissing interaction from those of secondary folding. The binding of metal ions to the small tertiary structure appeared to be saturable with an apparent Kd of 160 mM for K+ and 1.5 mM for Mg2+. The kissing formation was estimated to be associated with binding of ~2-3 diffuse K+ or Mg2+ ions. At their saturated binding, Mg2+ provided ~3 kcal/mol more stabilizing energy to the structure than K+. Furthermore, the cations change the unkissing forces significantly more than the kissing ones. For example, the presence of Mg2+ ions increased the average unkissing force from 21 pN to 44 pN, surprisingly high for breaking merely two base pairs; in contrast, the mean kissing force was changed by only 4.5 pN. Interestingly, the differential salt effects on the transition forces were not caused by different changes in the height of the kinetic barriers, but were instead attributed to how different molecular structures respond to the applied force. Our results showed the importance of diffuse cation binding to the stability of tertiary interaction and demonstrated the utility of mechanical unfolding in studying tertiary interactions.
DOI
Graded-index fiber tip optical tweezers: Numerical simulation and trapping experiment
Yuan Gong, Ai-Yan Ye, Yu Wu, Yun-Jiang Rao, Yao Yao, and Song Xiao
Optical fiber tweezers based on a graded-index multimode fiber (GIMMF) tip is proposed. Light propagation characteristics and gradient force distribution near the GIMMF tip are numerically investigated, which are further compared with that of optical fiber tips based on conventional single mode fibers. The simulated results indicated that by selecting optimal GIMMF length, the gradient force of the GIMMF tip tweezers is about 4 times higher than that of the SMF tip tweezers with a same shape. To prove the feasibility of such a new concept, optical trapping of yeast cells with a diameter of ~5 μm using the chemically-etched GIMMF tip is experimentally demonstrated and the trapping force is also calculated.
DOI
Optical fiber tweezers based on a graded-index multimode fiber (GIMMF) tip is proposed. Light propagation characteristics and gradient force distribution near the GIMMF tip are numerically investigated, which are further compared with that of optical fiber tips based on conventional single mode fibers. The simulated results indicated that by selecting optimal GIMMF length, the gradient force of the GIMMF tip tweezers is about 4 times higher than that of the SMF tip tweezers with a same shape. To prove the feasibility of such a new concept, optical trapping of yeast cells with a diameter of ~5 μm using the chemically-etched GIMMF tip is experimentally demonstrated and the trapping force is also calculated.
DOI
Calibration of spatial light modulators suffering from spatially varying phase response
David Engström, Martin Persson, Jörgen Bengtsson, and Mattias Goksör
We present a method for converting the desired phase values of a hologram to the correct pixel addressing values of a spatial light modulator (SLM), taking into account detailed spatial variations in the phase response of the SLM. In addition to thickness variations in the liquid crystal layer of the SLM, we also show that these variations in phase response can be caused by a non-uniform electric drive scheme in the SLM or by local heating caused by the incident laser beam. We demonstrate that the use of a global look-up table (LUT), even in combination with a spatially varying scale factor, generally does not yield sufficiently accurate conversion for applications requiring highly controllable output fields, such as holographic optical trapping (HOT). We therefore propose a method where the pixel addressing values are given by a three-dimensional polynomial, with two of the variables being the (x, y)-positions of the pixels, and the third their desired phase values. The coefficients of the polynomial are determined by measuring the phase response in 8×8 sub-sections of the SLM surface; the degree of the polynomial is optimized so that the polynomial expression nearly replicates the measurement in the measurement points, while still showing a good interpolation behavior in between. The polynomial evaluation increases the total computation time for hologram generation by only a few percent. Compared to conventional phase conversion methods, for an SLM with varying phase response, we found that the proposed method increases the control of the trap intensities in HOT, and efficiently prevents the appearance of strong unwanted 0th order diffraction that commonly occurs in SLM systems.
DOI
We present a method for converting the desired phase values of a hologram to the correct pixel addressing values of a spatial light modulator (SLM), taking into account detailed spatial variations in the phase response of the SLM. In addition to thickness variations in the liquid crystal layer of the SLM, we also show that these variations in phase response can be caused by a non-uniform electric drive scheme in the SLM or by local heating caused by the incident laser beam. We demonstrate that the use of a global look-up table (LUT), even in combination with a spatially varying scale factor, generally does not yield sufficiently accurate conversion for applications requiring highly controllable output fields, such as holographic optical trapping (HOT). We therefore propose a method where the pixel addressing values are given by a three-dimensional polynomial, with two of the variables being the (x, y)-positions of the pixels, and the third their desired phase values. The coefficients of the polynomial are determined by measuring the phase response in 8×8 sub-sections of the SLM surface; the degree of the polynomial is optimized so that the polynomial expression nearly replicates the measurement in the measurement points, while still showing a good interpolation behavior in between. The polynomial evaluation increases the total computation time for hologram generation by only a few percent. Compared to conventional phase conversion methods, for an SLM with varying phase response, we found that the proposed method increases the control of the trap intensities in HOT, and efficiently prevents the appearance of strong unwanted 0th order diffraction that commonly occurs in SLM systems.
DOI
Optical trapping and spectroscopic characterisation of ionic liquid solutions
Lee James Moore, Michael Summers and Grant Ritchie
This paper presents a study of the optical trapping of aerosols containing ionic liquid (IL). Droplets comprised of aqueous solutions of the IL ethylammonium nitrate (EAN) are demonstrated to be readily trapped by optical tweezers and are characterized spectroscopically, by analyising the morphology dependent resonances present within backscattered light originating from a broadband light emitting diode. The response of the droplets to conditions of varying relative humidity has also been investigated, as an important first step in measuring the uptake of gases by ILs. Finally, a comparison between broadband Mie scattering and cavity enhanced Raman spectroscopies is provided.
DOI
This paper presents a study of the optical trapping of aerosols containing ionic liquid (IL). Droplets comprised of aqueous solutions of the IL ethylammonium nitrate (EAN) are demonstrated to be readily trapped by optical tweezers and are characterized spectroscopically, by analyising the morphology dependent resonances present within backscattered light originating from a broadband light emitting diode. The response of the droplets to conditions of varying relative humidity has also been investigated, as an important first step in measuring the uptake of gases by ILs. Finally, a comparison between broadband Mie scattering and cavity enhanced Raman spectroscopies is provided.
DOI
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