Lars Friedrich and Alexander Rohrbach
For most optical tweezer applications, precise and reliable tracking of the trapped particle is an important requirement. Backfocal-plane interferometry is the fastest and most accurate tracking technique if the particle displacements are limited to half of the focal width. Especially for positive axial displacements, the nonlinear detector response can lead to incorrect tracking results. Here we show how the linear detection range around the trap center can be extended by a factor of 2 to 4 in the axial direction using a second frequency-detuned tracking focus that is generated by the same laser as the optical trap. Additionally, we show how the noise in the axial signal can be decreased significantly using a second detector.
DOI
Monday, May 31, 2010
Wavefront correction: Trapping through turbid media
Estela Martín-Badosa
The aberrations induced by strongly scattering and turbid samples make optical trapping in such media impossible. Now, researchers in Scotland have overcome the problem using in situ aberration correction.
The aberrations induced by strongly scattering and turbid samples make optical trapping in such media impossible. Now, researchers in Scotland have overcome the problem using in situ aberration correction.
Probing cytoplasmic organization and the actin cytoskeleton of plant cells with optical tweezers
Tijs Ketelaar, Hannie S. van der Honing and Anne Mie C. Emons
In interphase plant cells, the actin cytoskeleton is essential for intracellular transport and organization. To fully understand how the actin cytoskeleton functions as the structural basis for cytoplasmic organization, both molecular and physical aspects of the actin organization have to be considered. In the present review, we discuss literature that gives an insight into how cytoplasmic organization is achieved and in which actin-binding proteins have been identified that play a role in this process. We discuss how physical properties of the actin cytoskeleton in the cytoplasm of live plant cells, such as deformability and elasticity, can be probed by using optical tweezers. This technique allows non-invasive manipulation of cytoplasmic organization. Optical tweezers, integrated in a confocal microscope, can be used to manipulate cytoplasmic organization while studying actin dynamics. By combining this with mutant studies and drug applications, insight can be obtained about how the physical properties of the actin cytoskeleton, and thus the cytoplasmic organization, are influenced by different cellular processes.
DOI
In interphase plant cells, the actin cytoskeleton is essential for intracellular transport and organization. To fully understand how the actin cytoskeleton functions as the structural basis for cytoplasmic organization, both molecular and physical aspects of the actin organization have to be considered. In the present review, we discuss literature that gives an insight into how cytoplasmic organization is achieved and in which actin-binding proteins have been identified that play a role in this process. We discuss how physical properties of the actin cytoskeleton in the cytoplasm of live plant cells, such as deformability and elasticity, can be probed by using optical tweezers. This technique allows non-invasive manipulation of cytoplasmic organization. Optical tweezers, integrated in a confocal microscope, can be used to manipulate cytoplasmic organization while studying actin dynamics. By combining this with mutant studies and drug applications, insight can be obtained about how the physical properties of the actin cytoskeleton, and thus the cytoplasmic organization, are influenced by different cellular processes.
DOI
Thursday, May 27, 2010
Complex Fluids: Probing Mechanical Properties of Biological Systems with Optical Tweezers
H. Daniel Ou-Yang and Ming-Tzo Wei
The mechanical properties of cells are crucial for cell sensing and reaction to mechanical environments. This review describes the basic principles of optical tweezers and their use as force sensors for studying the mechanical properties of biological systems. It covers experiments of four groups of biological systems arranged by increasing complexity: (a) packaging DNA into viral capsids by bacteriophage portal motors and the dynamical stiffness of DNA upon protein binding, (b) actin-coated giant vesicles and the myosin-II embedded actin polymer network, (c) suspension cells, and (d) adhesion cells. These examples demonstrate how optical tweezers have been used to improve the understanding of the mechanical properties of biological systems at subcellular and molecular levels.
The mechanical properties of cells are crucial for cell sensing and reaction to mechanical environments. This review describes the basic principles of optical tweezers and their use as force sensors for studying the mechanical properties of biological systems. It covers experiments of four groups of biological systems arranged by increasing complexity: (a) packaging DNA into viral capsids by bacteriophage portal motors and the dynamical stiffness of DNA upon protein binding, (b) actin-coated giant vesicles and the myosin-II embedded actin polymer network, (c) suspension cells, and (d) adhesion cells. These examples demonstrate how optical tweezers have been used to improve the understanding of the mechanical properties of biological systems at subcellular and molecular levels.
Real-time optical micro-manipulation using optimized holograms generated on the GPU
S. Bianchi and R. Di Leonardo
Holographic optical tweezers allow the three-dimensional, dynamic, multipoint manipulation of micron sized objects using laser light. Exploiting the massive parallel architecture of modern GPUs we can generate highly optimized holograms at video frame-rate allowing the precise interactive micro-manipulation of complex structures.
3D Holographic Imaging and Trapping for Non-Invasive Cell Identification and Tracking
DaneshPanah, M., Zwick, S., Schaal, F., Warber, M., Javidi, B., Osten, W.
Real-time high-throughput identification, screening, characterization, and processing of biological specimen is of great interest to a host of areas spanning from cell biology and medicine to security and defense. Much like human biometrics, microorganisms exhibit natural signatures that can be used for identification. In this paper, we first overview two optical techniques, namely digital holographic microscopy and holographic optical tweezers which can non-invasively image, manipulate, and identify microorganisms in three dimensions. The two methods bear similarities in their optics and implementation. Thus, we have proposed a new approach to identification of micro/nano organisms and cells by combining the two methods of digital holographic microscopy and holographic optical tweezers which can be integrated into a single compact hardware. The proposed system can simultaneously sense, control, identify, and track cells and microorganisms in three dimensions. New possibilities that arise from the proposed method are discussed.
Real-time high-throughput identification, screening, characterization, and processing of biological specimen is of great interest to a host of areas spanning from cell biology and medicine to security and defense. Much like human biometrics, microorganisms exhibit natural signatures that can be used for identification. In this paper, we first overview two optical techniques, namely digital holographic microscopy and holographic optical tweezers which can non-invasively image, manipulate, and identify microorganisms in three dimensions. The two methods bear similarities in their optics and implementation. Thus, we have proposed a new approach to identification of micro/nano organisms and cells by combining the two methods of digital holographic microscopy and holographic optical tweezers which can be integrated into a single compact hardware. The proposed system can simultaneously sense, control, identify, and track cells and microorganisms in three dimensions. New possibilities that arise from the proposed method are discussed.
Wednesday, May 26, 2010
Change in spectrum of Brownian fluctuations of optically trapped red blood cells due to malarial infection
Vishal Saraogi, P. Padmapriya, Apurba Paul, Utpal S. Tatu, Vasant Natarajan
We study the properties of single red blood cells (RBCs) held in an optical-tweezers trap. We observe a change in the spectrum of Brownian fluctuations between RBCs from normal and malaria-infected samples. The change, caused by infection-induced structural changes in the cell, appears as a statistical increase in the mean (by 25%) and standard deviation (by 200%) of the corner frequency measured over ~100 cells. The increase is observed even though the ensemble of cells being measured consists mostly of cells that do not actually host the parasite, but are from an infected pool. This bystander effect appears to vindicate other observations that infected cells can affect the biomechanical properties of uninfected cells. The change is also observed to be independent of the stage of infection and its duration, highlighting its potential for disease detection.
We study the properties of single red blood cells (RBCs) held in an optical-tweezers trap. We observe a change in the spectrum of Brownian fluctuations between RBCs from normal and malaria-infected samples. The change, caused by infection-induced structural changes in the cell, appears as a statistical increase in the mean (by 25%) and standard deviation (by 200%) of the corner frequency measured over ~100 cells. The increase is observed even though the ensemble of cells being measured consists mostly of cells that do not actually host the parasite, but are from an infected pool. This bystander effect appears to vindicate other observations that infected cells can affect the biomechanical properties of uninfected cells. The change is also observed to be independent of the stage of infection and its duration, highlighting its potential for disease detection.
Bipolar optical forces on dielectric and metallic nanoparticles by evanescent wave: erratum
J. J. Xiao, H. H. Zheng, Y. X. Sun, and Y. Yao
A small error in a previous Letter [Opt. Lett.35, 962 (2010)OPLEDP0146-959210.1364/OL.35.000962] concerning the time-averaged Maxwell stress tensor is corrected here.
A small error in a previous Letter [Opt. Lett.35, 962 (2010)OPLEDP0146-959210.1364/OL.35.000962] concerning the time-averaged Maxwell stress tensor is corrected here.
Friday, May 21, 2010
Measurement of the Instantaneous Velocity of a Brownian Particle
Brownian motion of particles impacts many branches of science. We report on the Brownian motion of micron-sized beads of glass held in air in an optical tweezer, over a wide range of pressures, and measure the instantaneous velocity of a Brownian particle. Our results provide direct verification of the energy equipartition theorem for a Brownian particle. For short times, the ballistic regime of Brownian motion is observed, in contrast to the usual diffusive regime. We discuss the applications of these methods towards cooling the center of mass motion of a bead in vacuum to the quantum ground motional state.
Thursday, May 20, 2010
Hydrodynamic coupling in polygonal arrays of colloids: Experimental and analytical results
Giovanni M. Cicuta, Jurij Kotar, Aidan T. Brown, Ji-Ho Noh, and Pietro Cicuta
Colloidal particles are trapped harmonically on the vertices of planar regular polygons, using optical tweezers. The particles interact with each other via hydrodynamic coupling, which can be described adequately by Oseen’s tensor. Because of the interaction, the dynamics of any individual sphere is complex. Thermal motion results in a spectrum of relaxation times. The configuration of a system of N particles can be decomposed into 2N normal modes. In this work it is shown how to calculate these modes and their relaxation time scale analytically. The mathematical structure of the matrix of interaction leads to general properties for the symmetry of the normal modes and their dynamics, differing between the cases of even and odd N. The theory is compared to experiments performed on a range of rings with3≤N≤10, varying also the trap stiffness and the distance between particles.
Colloidal particles are trapped harmonically on the vertices of planar regular polygons, using optical tweezers. The particles interact with each other via hydrodynamic coupling, which can be described adequately by Oseen’s tensor. Because of the interaction, the dynamics of any individual sphere is complex. Thermal motion results in a spectrum of relaxation times. The configuration of a system of N particles can be decomposed into 2N normal modes. In this work it is shown how to calculate these modes and their relaxation time scale analytically. The mathematical structure of the matrix of interaction leads to general properties for the symmetry of the normal modes and their dynamics, differing between the cases of even and odd N. The theory is compared to experiments performed on a range of rings with3≤N≤10, varying also the trap stiffness and the distance between particles.
Visualizing the Formation and Collapse of DNA Toroids
Bram van den Broek, Maarten C. Noom, Joost van Mameren, Christopher Battle, Fred C. MacKintosh and Gijs J.L. Wuite
In living organisms, DNA is generally confined into very small volumes. In most viruses, positively charged multivalent ions assist the condensation of DNA into tightly packed toroidal structures. Interestingly, such cations can also induce the spontaneous formation of DNA toroids in vitro. To resolve the condensation dynamics and stability of DNA toroids, we use a combination of optical tweezers and fluorescence imaging to visualize in real-time spermine-induced (de)condensation in single DNA molecules. By actively controlling the DNA extension, we are able to follow (de)condensation under tension with high temporal and spatial resolution. We show that both processes occur in a quantized manner, caused by individual DNA loops added onto or removed from a toroidal condensate that is much smaller than previously observed in similar experiments. Finally, we present an analytical model that qualitatively captures the experimentally observed features, including an apparent force plateau.
Particle tracking stereomicroscopy in optical tweezers: Control of trap shape
Richard Bowman, Graham Gibson, and Miles Padgett
We present an optical system capable of generating stereoscopic images to track trapped particles in three dimensions. Two-dimensional particle tracking on each image yields three dimensional position information. Our approach allows the use of a high numerical aperture (NA= 1.3) objective and large separation angle, such that particles can be tracked axially with resolution of 3nm at 340Hz. Spatial Light Modulators (SLMs), the diffractive elements used to steer and split laser beams in Holographic Optical Tweezers, are also capable of more general operations. We use one here to vary the ratio of lateral to axial trap stiffness by changing the shape of the beam at the back aperture of the microscope objective. Beams which concentrate their optical power at the extremes of the back aperture give rise to much more efficient axial trapping. The flexibility of using an SLM allows us to create multiple traps with different shapes.
We present an optical system capable of generating stereoscopic images to track trapped particles in three dimensions. Two-dimensional particle tracking on each image yields three dimensional position information. Our approach allows the use of a high numerical aperture (NA= 1.3) objective and large separation angle, such that particles can be tracked axially with resolution of 3nm at 340Hz. Spatial Light Modulators (SLMs), the diffractive elements used to steer and split laser beams in Holographic Optical Tweezers, are also capable of more general operations. We use one here to vary the ratio of lateral to axial trap stiffness by changing the shape of the beam at the back aperture of the microscope objective. Beams which concentrate their optical power at the extremes of the back aperture give rise to much more efficient axial trapping. The flexibility of using an SLM allows us to create multiple traps with different shapes.
Two-Photon Quantum Dot Excitation during Optical Trapping
Liselotte Jauffred and Lene B. Oddershede
A single CW infrared laser beam can simultaneously trap and excite an individual colloidal quantum dot. Though the laser light is relatively weak, the excitation occurs through two-photon absorption. This finding eliminates the demand for an excitation light source in addition to a trapping laser in nanoscale experiments with simultaneous force-manipulation and quantum dot visualization. Also, we demonstrate that optical trapping efficiencies of individual quantum dots do not correlate with their emission wavelength or physical size.
A single CW infrared laser beam can simultaneously trap and excite an individual colloidal quantum dot. Though the laser light is relatively weak, the excitation occurs through two-photon absorption. This finding eliminates the demand for an excitation light source in addition to a trapping laser in nanoscale experiments with simultaneous force-manipulation and quantum dot visualization. Also, we demonstrate that optical trapping efficiencies of individual quantum dots do not correlate with their emission wavelength or physical size.
Optical-tweezer-induced microbubbles as scavengers of carbon nanotubes
Hema Ramachandran, A K Dharmadhikari, K Bambardekar, H Basu, J A Dharmadhikari, S Sharma and D Mathur
A modified optical tweezers set-up has been used to generate microbubbles in flowing, biologically relevant fluids and human whole blood that contains carbon nanotubes (CNTs) using low power (≤5 mW), infrared (1064 nm wavelength), continuous wave laser light. Temperature driven effects at the tweezers' focal point help to optically trap these microbubbles. It is observed that proximate CNTs are driven towards the focal spot where, on encountering the microbubble, they adhere to it. Such CNT-loaded microbubbles can be transported both along and against the flow of surrounding fluid, and can also be exploded to cause fragmentation of the bundles. Thus, microbubbles may be used for scavenging, transporting and dispersal of potentially toxic CNTs in biologically relevant environments.
A modified optical tweezers set-up has been used to generate microbubbles in flowing, biologically relevant fluids and human whole blood that contains carbon nanotubes (CNTs) using low power (≤5 mW), infrared (1064 nm wavelength), continuous wave laser light. Temperature driven effects at the tweezers' focal point help to optically trap these microbubbles. It is observed that proximate CNTs are driven towards the focal spot where, on encountering the microbubble, they adhere to it. Such CNT-loaded microbubbles can be transported both along and against the flow of surrounding fluid, and can also be exploded to cause fragmentation of the bundles. Thus, microbubbles may be used for scavenging, transporting and dispersal of potentially toxic CNTs in biologically relevant environments.
Measuring the Bending Stiffness of Bacterial Cells Using an Optical Trap
Siyuan Wang, Hugo Arellano-Santoyo, Peter A. Combs, Joshua W. Shaevitz
We developed a protocol to measure the bending rigidity of filamentous rod-shaped bacteria. Forces are applied with an optical trap, a microscopic three-dimensional spring made of light that is formed when a high-intensity laser beam is focused to a very small spot by a microscope's objective lens. To bend a cell, we first bind live bacteria to a chemically-treated coverslip. As these cells grow, the middle of the cells remains bound to the coverslip but the growing ends are free of this restraint. By inducing filamentous growth with the drug cephalexin, we are able to identify cells in which one end of the cell was stuck to the surface while the other end remained unattached and susceptible to bending forces. A bending force is then applied with an optical trap by binding a polylysine-coated bead to the tip of a growing cell. Both the force and the displacement of the bead are recorded and the bending stiffness of the cell is the slope of this relationship.
We developed a protocol to measure the bending rigidity of filamentous rod-shaped bacteria. Forces are applied with an optical trap, a microscopic three-dimensional spring made of light that is formed when a high-intensity laser beam is focused to a very small spot by a microscope's objective lens. To bend a cell, we first bind live bacteria to a chemically-treated coverslip. As these cells grow, the middle of the cells remains bound to the coverslip but the growing ends are free of this restraint. By inducing filamentous growth with the drug cephalexin, we are able to identify cells in which one end of the cell was stuck to the surface while the other end remained unattached and susceptible to bending forces. A bending force is then applied with an optical trap by binding a polylysine-coated bead to the tip of a growing cell. Both the force and the displacement of the bead are recorded and the bending stiffness of the cell is the slope of this relationship.
Wednesday, May 19, 2010
Holographic optical trapping of microrods and nanowires
Stephen H. Simpson and Simon Hanna
Holographic optical tweezing permits the trapping of objects with less than spherical symmetry in appropriately distributed sets of beams thereby permitting control to be exerted over both the orientation and position. In contrast to the familiar case of the singly trapped sphere, the stiffness and strength of such compound traps will have rotational components. We investigate this for a simple model system consisting of multiply trapped dielectric cylinder. Optically induced forces and torques are evaluated using the discrete dipole approximation and the resulting trap stiffnesses are presented. A variety of configurations of trapping beams are considered. Hydrodynamic resistances for the cylinder are also calculated and used to estimate translation and rotation rates. A number of conclusions are reached concerning the optimal trapping and dragging conditions for the rod. In particular, it is clear that it is advantageous to drag a rod in a direction perpendicular rather than parallel to its length. In addition, it is observed that the polarization of the incident light plays a significant role. Finally, it is noted that the non-conservative nature of the optical force field manifests itself directly in the stiffness of the trapped cylinder. The consequences of this last point are discussed.
Holographic optical tweezing permits the trapping of objects with less than spherical symmetry in appropriately distributed sets of beams thereby permitting control to be exerted over both the orientation and position. In contrast to the familiar case of the singly trapped sphere, the stiffness and strength of such compound traps will have rotational components. We investigate this for a simple model system consisting of multiply trapped dielectric cylinder. Optically induced forces and torques are evaluated using the discrete dipole approximation and the resulting trap stiffnesses are presented. A variety of configurations of trapping beams are considered. Hydrodynamic resistances for the cylinder are also calculated and used to estimate translation and rotation rates. A number of conclusions are reached concerning the optimal trapping and dragging conditions for the rod. In particular, it is clear that it is advantageous to drag a rod in a direction perpendicular rather than parallel to its length. In addition, it is observed that the polarization of the incident light plays a significant role. Finally, it is noted that the non-conservative nature of the optical force field manifests itself directly in the stiffness of the trapped cylinder. The consequences of this last point are discussed.
Monday, May 17, 2010
Optical forces on small magnetodielectric particle
M. Nieto-Vesperinas, J. J. Sáenz, R. Gómez-Medina, and L. Chantada
We present a study of the optical force on a small particle with both electric and magnetic response, immersed in an arbitrary non-absorbing medium, due to a generic incident electromagnetic field. Expressions for the gradient force, radiation pressure and curl components are obtained for the force due to both the electric and magnetic dipoles excited in the particle. In particular, for the magnetic force we tentatively introduce the concept of curl of the spin angular momentum density of the magnetic field, also expressed in terms of 3D generalizations of the Stokes parameters. From the formal analogy between the conservation of momentum and the optical theorem, we discuss the origin and significance of the self-interaction force between both dipoles; this is done in connection with that of the angular distribution of scattered light and of the extinction cross section.
We present a study of the optical force on a small particle with both electric and magnetic response, immersed in an arbitrary non-absorbing medium, due to a generic incident electromagnetic field. Expressions for the gradient force, radiation pressure and curl components are obtained for the force due to both the electric and magnetic dipoles excited in the particle. In particular, for the magnetic force we tentatively introduce the concept of curl of the spin angular momentum density of the magnetic field, also expressed in terms of 3D generalizations of the Stokes parameters. From the formal analogy between the conservation of momentum and the optical theorem, we discuss the origin and significance of the self-interaction force between both dipoles; this is done in connection with that of the angular distribution of scattered light and of the extinction cross section.
Direct observation of the myosin-Va power stroke and its reversal
James R Sellers, Claudia Veigel
Complex forms of cellular motility, including cell division, organelle trafficking or signal amplification in the auditory system, require strong coordination of the myosin motors involved. The most basic mechanism of coordination is via direct mechanical interactions of individual motor heads leading to modification of their mechanochemical cycles. Here we used an optical trap–based assay to investigate the reversibility of the force-generating conformational change (power stroke) of single myosin-Va motor heads. By applying load to the head shortly after binding to actin, we found that, at a certain load, the power stroke could be reversed, and the head fluctuated between an actin-bound pre– and a post–power stroke conformation. This load-dependent mechanical instability might be critical to coordinate the heads of processive, dimeric myosin-Va. Nonlinear response to load leading to coordination or oscillations amongst motors might be relevant for many cellular functions.
Complex forms of cellular motility, including cell division, organelle trafficking or signal amplification in the auditory system, require strong coordination of the myosin motors involved. The most basic mechanism of coordination is via direct mechanical interactions of individual motor heads leading to modification of their mechanochemical cycles. Here we used an optical trap–based assay to investigate the reversibility of the force-generating conformational change (power stroke) of single myosin-Va motor heads. By applying load to the head shortly after binding to actin, we found that, at a certain load, the power stroke could be reversed, and the head fluctuated between an actin-bound pre– and a post–power stroke conformation. This load-dependent mechanical instability might be critical to coordinate the heads of processive, dimeric myosin-Va. Nonlinear response to load leading to coordination or oscillations amongst motors might be relevant for many cellular functions.
Saturday, May 15, 2010
In situ wavefront correction and its application to micromanipulation
Tomáš Čižmár, Michael Mazilu & Kishan Dholakia
In any optical system, distortions to a propagating wavefront reduce the spatial coherence of a light field, making it increasingly difficult to obtain the theoretical diffraction-limited spot size. Such aberrations are severely detrimental to optimal performance in imaging, nanosurgery, nanofabrication and micromanipulation, as well as other techniques within modern microscopy. We present a generic method based on complex modulation for true in situ wavefront correction that allows compensation of all aberrations along the entire optical train. The power of the method is demonstrated for the field of micromanipulation, which is very sensitive to wavefront distortions. We present direct trapping with optimally focused laser light carrying power of a fraction of a milliwatt as well as the first trapping through highly turbid and diffusive media. This opens up new perspectives for optical micromanipulation in colloidal and biological physics and may be useful for various forms of advanced imaging.
In any optical system, distortions to a propagating wavefront reduce the spatial coherence of a light field, making it increasingly difficult to obtain the theoretical diffraction-limited spot size. Such aberrations are severely detrimental to optimal performance in imaging, nanosurgery, nanofabrication and micromanipulation, as well as other techniques within modern microscopy. We present a generic method based on complex modulation for true in situ wavefront correction that allows compensation of all aberrations along the entire optical train. The power of the method is demonstrated for the field of micromanipulation, which is very sensitive to wavefront distortions. We present direct trapping with optimally focused laser light carrying power of a fraction of a milliwatt as well as the first trapping through highly turbid and diffusive media. This opens up new perspectives for optical micromanipulation in colloidal and biological physics and may be useful for various forms of advanced imaging.
Friday, May 14, 2010
Optical trapping and manipulation of magnetic holes dispersed in a magnetic fluid
Ting Sun, Zhi-Cheng Fu, Wei-Ren Zhao, Hai-Dong Deng, Qiao-Feng Dai, Li-Jun Wu, Sheng Lan, and Achanta Venu Gopal
The optical trapping and manipulation of magnetic holes (MHs) dispersed in a magnetic fluid is systematically investigated. It is found that the gradient force, which tends to attract MHs to the beam center, can be completely counteracted by the repulsive force between MHs induced by a magnetic field. As a result, a depletion region is created at the laser beam spot for a sufficiently strong magnetic field. This phenomenon can be easily observed for large MHs with a diameter of 11 μm. However, it does not appear for MHs with a smaller diameter of 4.3 μm. It is revealed that the enhancement in the concentration of magnetic nanoparticles in the laser spot region as well as the clustering of these nanoparticles leads to a much stronger interaction between MHs when a magnetic field is applied. Consequently, the magnetic field strength necessary to create the depletion region is significantly reduced. We also find that the trapping behavior of MHs depends strongly on the thickness of the sample cells. For thin sample cells in which only one layer (or a two-dimensional distribution) of MHs is allowed, we can observe the creation of depletion region. In sharp contrast, MHs can be stably trapped at the center of the laser beam in thick sample cells even if a strong magnetic field is imposed. This phenomenon can be explained by the existence of a gradient in magnetic field strength along the direction perpendicular to the sample cells. Apart from individual MHs, we also investigate the movement of MH chains under the scattering force of the laser beam. It is observed that MH chains always move along the direction parallel to the magnetic field. This behavior can be easily understood when the anisotropy in viscosity caused by the applied magnetic field is considered.
The optical trapping and manipulation of magnetic holes (MHs) dispersed in a magnetic fluid is systematically investigated. It is found that the gradient force, which tends to attract MHs to the beam center, can be completely counteracted by the repulsive force between MHs induced by a magnetic field. As a result, a depletion region is created at the laser beam spot for a sufficiently strong magnetic field. This phenomenon can be easily observed for large MHs with a diameter of 11 μm. However, it does not appear for MHs with a smaller diameter of 4.3 μm. It is revealed that the enhancement in the concentration of magnetic nanoparticles in the laser spot region as well as the clustering of these nanoparticles leads to a much stronger interaction between MHs when a magnetic field is applied. Consequently, the magnetic field strength necessary to create the depletion region is significantly reduced. We also find that the trapping behavior of MHs depends strongly on the thickness of the sample cells. For thin sample cells in which only one layer (or a two-dimensional distribution) of MHs is allowed, we can observe the creation of depletion region. In sharp contrast, MHs can be stably trapped at the center of the laser beam in thick sample cells even if a strong magnetic field is imposed. This phenomenon can be explained by the existence of a gradient in magnetic field strength along the direction perpendicular to the sample cells. Apart from individual MHs, we also investigate the movement of MH chains under the scattering force of the laser beam. It is observed that MH chains always move along the direction parallel to the magnetic field. This behavior can be easily understood when the anisotropy in viscosity caused by the applied magnetic field is considered.
Motor Coordination via a Tug-of-War Mechanism Drives Bidirectional Vesicle Transport
Adam G. Hendricks, Eran Perlson, Jennifer L. Ross, Harry W. Schroeder III, Mariko Tokito and Erika L.F. Holzbaur
The microtubule motors kinesin and dynein function collectively to drive vesicular transport. High-resolution tracking of vesicle motility in the cell indicates that transport is often bidirectional, characterized by frequent directional changes. However, the mechanisms coordinating the collective activities of oppositely oriented motors bound to the same cargo are not well understood. To examine motor coordination, we purified neuronal transport vesicles and analyzed their motility via automated particle tracking with nanometer resolution. The motility of purified vesicles reconstituted in vitro closely models the movement of LysoTracker-positive vesicles in primary neurons, where processive bidirectional motility is interrupted with frequent directional switches, diffusional movement, and pauses. Quantitative analysis indicates that vesicles copurify with a low number of stably bound motors: one to five dynein and one to four kinesin motors. These observations compare well to predictions from a stochastic tug-of-war model, where transport is driven by the force-dependent kinetics of teams of opposing motors in the absence of external regulation. Together, these observations indicate that vesicles move robustly with a small complement of tightly bound motors and suggest an efficient regulatory scheme for bidirectional motility where small changes in the number of engaged motors manifest in large changes in the motility of cargo.
The microtubule motors kinesin and dynein function collectively to drive vesicular transport. High-resolution tracking of vesicle motility in the cell indicates that transport is often bidirectional, characterized by frequent directional changes. However, the mechanisms coordinating the collective activities of oppositely oriented motors bound to the same cargo are not well understood. To examine motor coordination, we purified neuronal transport vesicles and analyzed their motility via automated particle tracking with nanometer resolution. The motility of purified vesicles reconstituted in vitro closely models the movement of LysoTracker-positive vesicles in primary neurons, where processive bidirectional motility is interrupted with frequent directional switches, diffusional movement, and pauses. Quantitative analysis indicates that vesicles copurify with a low number of stably bound motors: one to five dynein and one to four kinesin motors. These observations compare well to predictions from a stochastic tug-of-war model, where transport is driven by the force-dependent kinetics of teams of opposing motors in the absence of external regulation. Together, these observations indicate that vesicles move robustly with a small complement of tightly bound motors and suggest an efficient regulatory scheme for bidirectional motility where small changes in the number of engaged motors manifest in large changes in the motility of cargo.
Optofluidic chip for single cell trapping and stretching fabricated by a femtosecond laser
Francesca Bragheri, Lorenzo Ferrara, Nicola Bellini, Krishna C. Vishnubhatla, Paolo Minzioni, Roberta Ramponi, Roberto Osellame, Ilaria Cristiani
The authors present the design and optimization of an optofluidic monolithic chip, able to provide optical trapping and controlled stretching of single cells. The chip is fabricated in a fused silica glass substrate by femtosecond laser micromachining which can produce both optical waveguides and microfluidic channels with great accuracy. A new fabrication procedure adopted in this work allows the demonstration of microchannels with a square cross-section, thus guaranteeing an improved quality of the trapped cell images. Femtosecond laser micromachining emerges as a promising technique for the development of multifunctional integrated biophotonic devices that can be easily coupled to a microscope platform, thus enabling a complete characterization of the cells under test.
The authors present the design and optimization of an optofluidic monolithic chip, able to provide optical trapping and controlled stretching of single cells. The chip is fabricated in a fused silica glass substrate by femtosecond laser micromachining which can produce both optical waveguides and microfluidic channels with great accuracy. A new fabrication procedure adopted in this work allows the demonstration of microchannels with a square cross-section, thus guaranteeing an improved quality of the trapped cell images. Femtosecond laser micromachining emerges as a promising technique for the development of multifunctional integrated biophotonic devices that can be easily coupled to a microscope platform, thus enabling a complete characterization of the cells under test.
Optical manipulation for single-cell studies
Kerstin Ramser, Dag Hanstorp
In the last decade optical manipulation has evolved from a field of interest for physicists to a versatile tool widely used within life sciences. This has been made possible in particular due to the development of a large variety of imaging techniques that allow detailed information to be gained from investigations of single cells. The use of multiple optical traps has high potential within single-cell analysis since parallel measurements provide good statistics. Multifunctional optical tweezers are, for instance, used to study cell heterogeneity in an ensemble, and force measurements are used to investigate the mechanical properties of individual cells. Investigations of molecular motors and forces on the single-molecule level have led to discoveries that would have been difficult to make with other techniques. Optical manipulation has prospects within the field of cell signalling and tissue engineering. When combined with microfluidic systems the chemical environment of cells can be precisely controlled. Hence the influence of pH, salt concentration, drugs and temperature can be investigated in real time. Fast advancing technical developments of automated and user-friendly optical manipulation tools and cross-disciplinary collaboration will contribute to the routinely use of optical manipulation techniques within the life sciences.
Thursday, May 13, 2010
Mueller matrix-based optimization of reflective type twisted nematic liquid crystal SLM at oblique incidences
R.S. Verma, M.K. Swami, S.S. Manhas and P.K. Gupta
Mueller matrix measurements were used to characterize the polarization properties of liquid crystal-based reflective type twisted nematic (TN) special light modulator (SLM) at oblique incidence of the laser beam. The experimentally obtained Mueller matrices were used to obtain the combination of polarization optics required to optimize it for phase only modulation. The results indicate that minimum intensity modulation is obtained with the use of a polarizer followed by a quarter wave plate (QWP) in polarization state generator (PSG) arm and a QWP followed by an analyzer in polarization state analyzer arm (PSA). Polarization parameters such as retardance, rotation and depolarization were calculated from the experimentally obtained Mueller matrices using polar decomposition method at different angle of incidences of the laser beam and the results has been discussed. The similarity between retardance and depolarization curve as a function of address voltage of TNSLM indicated that depolarization is mainly associated with errors in retardance values. Further, spectral Mueller matrix measurements were used to obtain intensity modulation response in the range of wavelengths 450–700 nm for broadband applications.
Mueller matrix measurements were used to characterize the polarization properties of liquid crystal-based reflective type twisted nematic (TN) special light modulator (SLM) at oblique incidence of the laser beam. The experimentally obtained Mueller matrices were used to obtain the combination of polarization optics required to optimize it for phase only modulation. The results indicate that minimum intensity modulation is obtained with the use of a polarizer followed by a quarter wave plate (QWP) in polarization state generator (PSG) arm and a QWP followed by an analyzer in polarization state analyzer arm (PSA). Polarization parameters such as retardance, rotation and depolarization were calculated from the experimentally obtained Mueller matrices using polar decomposition method at different angle of incidences of the laser beam and the results has been discussed. The similarity between retardance and depolarization curve as a function of address voltage of TNSLM indicated that depolarization is mainly associated with errors in retardance values. Further, spectral Mueller matrix measurements were used to obtain intensity modulation response in the range of wavelengths 450–700 nm for broadband applications.
Tunable gradient force of hyperbolic-cosine–Gaussian beam with vortices
Xiumin Gao, Zhuo Li, Jian Wang, Lingling Sun and Songlin Zhuang
Gradient force plays an important role in optical tweezers technique. In this paper, the tunable gradient force in focal plane of the hyperbolic-cosine–Gaussian (ChG) beam is investigated numerically. The ChG beam contains one spiral vortex and one non-spiral vortex. Simulation results show that the gradient force distribution can be altered considerably by decentered parameters of ChG beam, topological number of the spiral vortex, and vortex parameter of the non-spiral vortex. Many novel gradient force patterns can occur, which means corresponding optical traps may come into being, including ring optical trap, multiple-point trap pattern, line optical trap, rectangle trap pattern, and rhombus trap pattern. In addition, force pattern evolution principle may also differ significantly.
Gradient force plays an important role in optical tweezers technique. In this paper, the tunable gradient force in focal plane of the hyperbolic-cosine–Gaussian (ChG) beam is investigated numerically. The ChG beam contains one spiral vortex and one non-spiral vortex. Simulation results show that the gradient force distribution can be altered considerably by decentered parameters of ChG beam, topological number of the spiral vortex, and vortex parameter of the non-spiral vortex. Many novel gradient force patterns can occur, which means corresponding optical traps may come into being, including ring optical trap, multiple-point trap pattern, line optical trap, rectangle trap pattern, and rhombus trap pattern. In addition, force pattern evolution principle may also differ significantly.
Characterization of Bacterial Spore Germination Using Integrated Phase Contrast Microscopy, Raman Spectroscopy, and Optical Tweezers
Lingbo Kong, Pengfei Zhang, Peter Setlow and Yong-qing Li
We present a methodology that combines external phase contrast microscopy, Raman spectroscopy, and optical tweezers to monitor a variety of changes during the germination of single Bacillus cereus spores in both nutrient (l-alanine) and non-nutrient (Ca-dipicolinic acid (DPA)) germinants with a temporal resolution of 2 s. Phase contrast microscopy assesses changes in refractility of individual spores during germination, while Raman spectroscopy gives information on changes in spore-specific molecules. The results obtained include (1) the brightness of the phase contrast image of an individual dormant spore is proportional to the level of CaDPA in that spore; (2) the end of the first Stage of germination, revealed as the end of the rapid drop in spore refractility by phase contrast microscopy, precisely corresponds to the completion of the release of CaDPA as revealed by Raman spectroscopy; and (3) the correspondence between the rapid drop in spore refractility and complete CaDPA release was observed not only for spores germinating in the well-controlled environment of an optical trap but also for spores germinating when adhered on a microscope coverslip. Using this latter method, we also simultaneously characterized the distribution of the time-to-complete-CaDPA release (Trelease) of hundreds of individual B. cereus spores germinating with both saturating and subsaturating concentrations of l-alanine and with CaDPA.
We present a methodology that combines external phase contrast microscopy, Raman spectroscopy, and optical tweezers to monitor a variety of changes during the germination of single Bacillus cereus spores in both nutrient (l-alanine) and non-nutrient (Ca-dipicolinic acid (DPA)) germinants with a temporal resolution of 2 s. Phase contrast microscopy assesses changes in refractility of individual spores during germination, while Raman spectroscopy gives information on changes in spore-specific molecules. The results obtained include (1) the brightness of the phase contrast image of an individual dormant spore is proportional to the level of CaDPA in that spore; (2) the end of the first Stage of germination, revealed as the end of the rapid drop in spore refractility by phase contrast microscopy, precisely corresponds to the completion of the release of CaDPA as revealed by Raman spectroscopy; and (3) the correspondence between the rapid drop in spore refractility and complete CaDPA release was observed not only for spores germinating in the well-controlled environment of an optical trap but also for spores germinating when adhered on a microscope coverslip. Using this latter method, we also simultaneously characterized the distribution of the time-to-complete-CaDPA release (Trelease) of hundreds of individual B. cereus spores germinating with both saturating and subsaturating concentrations of l-alanine and with CaDPA.
Influence of Noise on Force Measurements
Giovanni Volpe, Laurent Helden, Thomas Brettschneider, Jan Wehr, and Clemens Bechinger
We demonstrate how the ineluctable presence of thermal noise alters the measurement of forces acting on microscopic and nanoscopic objects. We quantify this effect exemplarily for a Brownian particle near a wall subjected to gravitational and electrostatic forces. Our results demonstrate that the force-measurement process is prone to artifacts if the noise is not correctly taken into account.
We demonstrate how the ineluctable presence of thermal noise alters the measurement of forces acting on microscopic and nanoscopic objects. We quantify this effect exemplarily for a Brownian particle near a wall subjected to gravitational and electrostatic forces. Our results demonstrate that the force-measurement process is prone to artifacts if the noise is not correctly taken into account.
Minimizing intensity fluctuations in dynamic holographic optical tweezers by restricted phase change
Martin Persson, David Engström, Anders Frank, Jan Backsten, Jörgen Bengtsson, and Mattias Goksör
We present a method for reducing intensity fluctuations that typically occur when a spatial light modulator is updated between consecutive computer generated holograms. The method is applicable to most iterative hologram generating algorithms and minimizes the average phase difference between consecutive holograms. Applications with high stability requirements, such as optical force measurement with holographic optical tweezers, should benefit from this improvement.
Generalised phase contrast: microscopy, manipulation and more
Darwin Palima, Jesper Glückstad
Generalised phase contrast (GPC) not only leads to more accurate phase imaging beyond thin biological samples, but serves as an enabling framework in developing tools over a wide spectrum of contemporary applications in optics and photonics, including optical trapping and micromanipulation, optical phase cryptography, light-efficient image projection and parallel laser beam shaping for optical landscapes. In this review, we discuss the fundamental ideas behind generalised phase contrast and present a survey of its exciting applications.
Generalised phase contrast (GPC) not only leads to more accurate phase imaging beyond thin biological samples, but serves as an enabling framework in developing tools over a wide spectrum of contemporary applications in optics and photonics, including optical trapping and micromanipulation, optical phase cryptography, light-efficient image projection and parallel laser beam shaping for optical landscapes. In this review, we discuss the fundamental ideas behind generalised phase contrast and present a survey of its exciting applications.
Characterization of Lactobacillus reuteri Interaction with Milk Fat Globule Membrane Components in Dairy Products
Guillaume Brisson, Hannah F. Payken, John P. Sharpe and Rafael Jiménez-Flores
A set of methods has been developed to study the adhesion between four Lactobacillus reuteri strains and the milk fat globule membrane (MFGM) components in dairy products. By combining sucrose density gradient (SDG) centrifugation and bacterial DNA quantification it was found which strains of L. reuteri were more strongly associated with the dairy products, and the results were corroborated by direct binding rate and force measurements made with optical tweezers. It was determined that strong binding was associated with hydrophobicity of the bacteria and that this hydrophobicity is correlated with the presence of LiCl-extractable protein on the surface of the bacteria. Confocal laser scanning microscopy (CLSM) allowed for the visualization of interactions between bacteria and MFGM. This study demonstrates that these methods can be used in combination to characterize, both qualitatively and quantitatively, the adhesion of lactic acid bacteria strains in dairy products.
A set of methods has been developed to study the adhesion between four Lactobacillus reuteri strains and the milk fat globule membrane (MFGM) components in dairy products. By combining sucrose density gradient (SDG) centrifugation and bacterial DNA quantification it was found which strains of L. reuteri were more strongly associated with the dairy products, and the results were corroborated by direct binding rate and force measurements made with optical tweezers. It was determined that strong binding was associated with hydrophobicity of the bacteria and that this hydrophobicity is correlated with the presence of LiCl-extractable protein on the surface of the bacteria. Confocal laser scanning microscopy (CLSM) allowed for the visualization of interactions between bacteria and MFGM. This study demonstrates that these methods can be used in combination to characterize, both qualitatively and quantitatively, the adhesion of lactic acid bacteria strains in dairy products.
Dynamics of a trapped Brownian particle in shear flows
Lukas Holzer, Jochen Bammert, Roland Rzehak, and Walter Zimmermann
The Brownian motion of a particle in a harmonic potential, which is simultaneously exposed either to a linear shear flow or to a plane Poiseuille flow is investigated. In the shear plane of both flows the probability distribution of the particle becomes anisotropic and the dynamics is changed in a characteristic manner compared to a trapped particle in a quiescent fluid. The particle distribution takes either an elliptical or a parachute shape or a superposition of both depending on the mean particle position in the shear plane. Simultaneously, shear-induced cross-correlations between particle fluctuations along orthogonal directions in the shear plane are found. They are asymmetric in time. In Poiseuille flow thermal particle fluctuations perpendicular to the flow direction in the shear plane induce a shift of the particle’s mean position away from the potential minimum. Two complementary methods are suggested to measure shear-induced cross-correlations between particle fluctuations along orthogonal directions.
The Brownian motion of a particle in a harmonic potential, which is simultaneously exposed either to a linear shear flow or to a plane Poiseuille flow is investigated. In the shear plane of both flows the probability distribution of the particle becomes anisotropic and the dynamics is changed in a characteristic manner compared to a trapped particle in a quiescent fluid. The particle distribution takes either an elliptical or a parachute shape or a superposition of both depending on the mean particle position in the shear plane. Simultaneously, shear-induced cross-correlations between particle fluctuations along orthogonal directions in the shear plane are found. They are asymmetric in time. In Poiseuille flow thermal particle fluctuations perpendicular to the flow direction in the shear plane induce a shift of the particle’s mean position away from the potential minimum. Two complementary methods are suggested to measure shear-induced cross-correlations between particle fluctuations along orthogonal directions.
Tuesday, May 11, 2010
Optical trapping of micrometer-sized dielectric particles by cylindrical vector beams
Yuichi Kozawa and Shunichi Sato
Single-beam optical trapping of micrometer-sized dielectric particles is experimentally demonstrated using radially and azimuthally polarized beams. The axial and transverse optical trapping efficiencies of glass and polystyrene beads suspended in water are measured. The radially polarized beam exhibited the highest trapping efficiency in the axial direction due to the p polarization of the radial polarization on the particle surface. On the other hand, the azimuthally polarized beam had a higher transverse trapping efficiency than the radially polarized beam. These results are consistent with numerical predictions.
Cytoplasmic dynein is not a conventional processive motor
Wilhelm J. Walter, Bernhard Brenner and Walter Steffen
Cytoplasmic dynein is a microtubule-based molecular motor with a multitude of functions from cell division to organelle transport. Cargo transport is often achieved as a co-complex with dynactin and it is believed that this co-complex enhances the processive translocation of cargo along the microtubule tracks ([King and Schroer, 2000] and [Culver-Hanlon et al., 2006]). Single molecule studies have revealed that dynein on its own can also act as a processive motor (Reck-Peterson et al., 2006; Toba et al., 2006). However, these studies did not allow the detection of a non-processive motor function. Previous studies based on the transport of vesicles or liposomes indicated that processive transport could only be achieved by an ensemble of motor molecules (Schroer & Sheetz, 1991; [Wang and Sheetz, 2000] and [Muresan et al., 2001]).Here we use the three bead dumbbell assay to show for the first time, that cytoplasmic dynein is a non-processive motor at low ATP concentrations. Processivity can be restored even in the absence of dynactin by increasing the ATP concentration to 100 μM. We propose that an altered occupancy of the different ATP binding sites (AAA1–4) acts as a modulator between processive and non-processive stepping.
Cytoplasmic dynein is a microtubule-based molecular motor with a multitude of functions from cell division to organelle transport. Cargo transport is often achieved as a co-complex with dynactin and it is believed that this co-complex enhances the processive translocation of cargo along the microtubule tracks ([King and Schroer, 2000] and [Culver-Hanlon et al., 2006]). Single molecule studies have revealed that dynein on its own can also act as a processive motor (Reck-Peterson et al., 2006; Toba et al., 2006). However, these studies did not allow the detection of a non-processive motor function. Previous studies based on the transport of vesicles or liposomes indicated that processive transport could only be achieved by an ensemble of motor molecules (Schroer & Sheetz, 1991; [Wang and Sheetz, 2000] and [Muresan et al., 2001]).Here we use the three bead dumbbell assay to show for the first time, that cytoplasmic dynein is a non-processive motor at low ATP concentrations. Processivity can be restored even in the absence of dynactin by increasing the ATP concentration to 100 μM. We propose that an altered occupancy of the different ATP binding sites (AAA1–4) acts as a modulator between processive and non-processive stepping.
Monday, May 10, 2010
Optical Tweezers as a Probe for Oligodeoxyribonucleotide Structuration
Nicola Borbone; Giorgia Oliviero; Jussara Amato; Gennaro Piccialli; Luciano Mayol; Lisa Miccio; Giuseppe Pesce; Antonio Sasso
The aim of this work is to investigate if the optical tweezers (OT) are suitable as a diagnostic tool for monitoring the oligodeoxyribonucleotide (ODN) structural behavior in solution. Preliminary experiments, performed on the quadruplex formed by the ODN sequence TGGGGT, showed that the OT can be used as a probe for ODN structuration by monitoring the medium viscosity changes associated with ODN folding-unfolding processes.
Analytical partial wave expansion of vector Bessel beam and its application to optical binding
Jun Chen, Jack Ng, Pei Wang, and Zhifang Lin
Explicit partial wave coefficients are derived for nondiffractive vector Bessel beam of arbitrary order and polarization. Calculations based on the analytical partial wave expansion yield results that agree well with recent numerical calculations and experiments on optical binding. We have also investigated how one can tailor the interparticle interaction by overlaying different coatings. It is found that the Ag and low dielectric coating can increase the number of equilibrium positions, whereas the antireflection coating reduces it.
Explicit partial wave coefficients are derived for nondiffractive vector Bessel beam of arbitrary order and polarization. Calculations based on the analytical partial wave expansion yield results that agree well with recent numerical calculations and experiments on optical binding. We have also investigated how one can tailor the interparticle interaction by overlaying different coatings. It is found that the Ag and low dielectric coating can increase the number of equilibrium positions, whereas the antireflection coating reduces it.
Chromatin Fiber Dynamics under Tension and Torsion
Christophe Lavelle, Jean-Marc Victor and Jordanka Zlatanova
Genetic and epigenetic information in eukaryotic cells is carried on chromosomes, basically consisting of large compact supercoiled chromatin fibers. Micromanipulations have recently led to great advances in the knowledge of the complex mechanisms underlying the regulation of DNA transaction events by nucleosome and chromatin structural changes. Indeed, magnetic and optical tweezers have allowed opportunities to handle single nucleosomal particles or nucleosomal arrays and measure their response to forces and torques, mimicking the molecular constraints imposed in vivo by various molecular motors acting on the DNA. These challenging technical approaches provide us with deeper understanding of the way chromatin dynamically packages our genome and participates in the regulation of cellular metabolism.
Genetic and epigenetic information in eukaryotic cells is carried on chromosomes, basically consisting of large compact supercoiled chromatin fibers. Micromanipulations have recently led to great advances in the knowledge of the complex mechanisms underlying the regulation of DNA transaction events by nucleosome and chromatin structural changes. Indeed, magnetic and optical tweezers have allowed opportunities to handle single nucleosomal particles or nucleosomal arrays and measure their response to forces and torques, mimicking the molecular constraints imposed in vivo by various molecular motors acting on the DNA. These challenging technical approaches provide us with deeper understanding of the way chromatin dynamically packages our genome and participates in the regulation of cellular metabolism.
Friday, May 7, 2010
Laser induced rotation of trapped chiral and achiral nematic droplets
Marjan Mosallaeipour; Yashodhan Hatwalne; N. V. Madhusudana; Sharath Ananthamurthy
We study the response of optically trapped achiral and chiralized nematic liquid crystal droplets to linear as well as circular polarized light. We find that there is internal dissipation in rotating achiral nematic droplets trapped in glycerine. We also demonstrate that some chiralized droplets rotate under linearly polarized light. The best fit to our data on chiralized droplets indicates that rotational frequency of these droplets with radius R is approximately proportional to 1/R2, rather than to 1/R3.
We study the response of optically trapped achiral and chiralized nematic liquid crystal droplets to linear as well as circular polarized light. We find that there is internal dissipation in rotating achiral nematic droplets trapped in glycerine. We also demonstrate that some chiralized droplets rotate under linearly polarized light. The best fit to our data on chiralized droplets indicates that rotational frequency of these droplets with radius R is approximately proportional to 1/R2, rather than to 1/R3.
Proliferation of anomalous symmetries in colloidal monolayers subjected to quasiperiodic light fields
Jules Mikhael, Michael Schmiedeberg, Sebastian Rausch, Johannes Roth, Holger Stark, and Clemens Bechinger
Quasicrystals provide a fascinating class of materials with intriguing properties. Despite a strong potential for numerous technical applications, the conditions under which quasicrystals form are still poorly understood. Currently, it is not clear why most quasicrystals hold 5- or 10-fold symmetry but no single example with 7- or 9-fold symmetry has ever been observed. Here we report on geometrical constraints which impede the formation of quasicrystals with certain symmetries in a colloidal model system. Experimentally, colloidal quasicrystals are created by subjecting micron-sized particles to two-dimensional quasiperiodic potential landscapes created by n = 5 or seven laser beams. Our results clearly demonstrate that quasicrystalline order is much easier established for n = 5 compared to n = 7. With increasing laser intensity we observe that the colloids first adopt quasiperiodic order at local areas which then laterally grow until an extended quasicrystalline layer forms. As nucleation sites where quasiperiodicity originates, we identify highly symmetric motifs in the laser pattern. We find that their density strongly varies with n and surprisingly is smallest exactly for those quasicrystalline symmetries which have never been observed in atomic systems. Since such high-symmetry motifs also exist in atomic quasicrystals where they act as preferential adsorption sites, this suggests that it is indeed the deficiency of such motifs which accounts for the absence of materials with e.g., 7-fold symmetry.
Quasicrystals provide a fascinating class of materials with intriguing properties. Despite a strong potential for numerous technical applications, the conditions under which quasicrystals form are still poorly understood. Currently, it is not clear why most quasicrystals hold 5- or 10-fold symmetry but no single example with 7- or 9-fold symmetry has ever been observed. Here we report on geometrical constraints which impede the formation of quasicrystals with certain symmetries in a colloidal model system. Experimentally, colloidal quasicrystals are created by subjecting micron-sized particles to two-dimensional quasiperiodic potential landscapes created by n = 5 or seven laser beams. Our results clearly demonstrate that quasicrystalline order is much easier established for n = 5 compared to n = 7. With increasing laser intensity we observe that the colloids first adopt quasiperiodic order at local areas which then laterally grow until an extended quasicrystalline layer forms. As nucleation sites where quasiperiodicity originates, we identify highly symmetric motifs in the laser pattern. We find that their density strongly varies with n and surprisingly is smallest exactly for those quasicrystalline symmetries which have never been observed in atomic systems. Since such high-symmetry motifs also exist in atomic quasicrystals where they act as preferential adsorption sites, this suggests that it is indeed the deficiency of such motifs which accounts for the absence of materials with e.g., 7-fold symmetry.
Thursday, May 6, 2010
Characterization of hydrogel microstructure using laser tweezers particle tracking and confocal reflection imaging
M A Kotlarchyk, E L Botvinick and A J Putnam
Hydrogels are commonly used as extracellular matrix mimetics for applications in tissue engineering and increasingly as cell culture platforms with which to study the influence of biophysical and biochemical cues on cell function in 3D. In recent years, a significant number of studies have focused on linking substrate mechanical properties to cell function using standard methodologies to characterize the bulk mechanical properties of the hydrogel substrates. However, current understanding of the correlations between the microstructural mechanical properties of hydrogels and cell function in 3D is poor, in part because of a lack of appropriate techniques. Here we have utilized a laser tracking system, based on passive optical microrheology instrumentation, to characterize the microstructure of viscoelastic fibrin clots. Trajectories and mean square displacements were observed as bioinert PEGylated (PEG: polyethylene glycol) microspheres (1, 2 or 4.7 µm in diameter) diffused within confined pores created by the protein phase of fibrin hydrogels. Complementary confocal reflection imaging revealed microstructures comprised of a highly heterogeneous fibrin network with a wide range of pore sizes. As the protein concentration of fibrin gels was increased, our quantitative laser tracking measurements showed a corresponding decrease in particle mean square displacements with greater resolution and sensitivity than conventional imaging techniques. This platform-independent method will enable a more complete understanding of how changes in substrate mechanical properties simultaneously influence other microenvironmental parameters in 3D cultures.
E. coli NusG Inhibits Backtracking and Accelerates Pause-Free Transcription by Promoting Forward Translocation of RNA Polymerase
Kristina M. Herbert Jing Zhou, Rachel A. Mooney, Arthur La Porta, Robert Landick and Steven M. Block
NusG is an essential transcription factor in Escherichia coli that is capable of increasing the overall rate of transcription. Transcript elongation by RNA polymerase (RNAP) is frequently interrupted by pauses of varying durations, and NusG is known to decrease the occupancy of at least some paused states. However, it has not been established whether NusG enhances transcription chiefly by (1) increasing the rate of elongation between pauses, (2) reducing the lifetimes of pauses, or (3) reducing the rate of entry into paused states. Here, we studied transcription by single molecules of RNAP under various conditions of ribonucleoside triphosphate concentration, applied load, and temperature, using an optical trapping assay capable of distinguishing pauses as brief as 1 s. We found that NusG increases the rate of elongation, that is, the pause-free velocity along the template. Because pauses are off-pathway states that compete with elongation, we observed a concomitant decrease in the rate of entry into short-lifetime, paused states. The effects on short pauses and elongation were comparatively modest, however. More dramatic was the effect of NusG on suppressing entry into long-lifetime (“stabilized”) pauses. Because a significant fraction of the time required for the transcription of a typical gene may be occupied by long pauses, NusG is capable of exerting a significant modulatory effect on the rates of RNA synthesis. The observed properties of NusG were consistent with a unified model where the function of this accessory factor is to promote transcriptionally downstream motion of the enzyme along the DNA template, which has the effect of forward-biasing RNAP from the pre-translocated state toward the post-translocated state.
NusG is an essential transcription factor in Escherichia coli that is capable of increasing the overall rate of transcription. Transcript elongation by RNA polymerase (RNAP) is frequently interrupted by pauses of varying durations, and NusG is known to decrease the occupancy of at least some paused states. However, it has not been established whether NusG enhances transcription chiefly by (1) increasing the rate of elongation between pauses, (2) reducing the lifetimes of pauses, or (3) reducing the rate of entry into paused states. Here, we studied transcription by single molecules of RNAP under various conditions of ribonucleoside triphosphate concentration, applied load, and temperature, using an optical trapping assay capable of distinguishing pauses as brief as 1 s. We found that NusG increases the rate of elongation, that is, the pause-free velocity along the template. Because pauses are off-pathway states that compete with elongation, we observed a concomitant decrease in the rate of entry into short-lifetime, paused states. The effects on short pauses and elongation were comparatively modest, however. More dramatic was the effect of NusG on suppressing entry into long-lifetime (“stabilized”) pauses. Because a significant fraction of the time required for the transcription of a typical gene may be occupied by long pauses, NusG is capable of exerting a significant modulatory effect on the rates of RNA synthesis. The observed properties of NusG were consistent with a unified model where the function of this accessory factor is to promote transcriptionally downstream motion of the enzyme along the DNA template, which has the effect of forward-biasing RNAP from the pre-translocated state toward the post-translocated state.
3D multiple optical trapping of Au-nanoparticles and prokaryote E. coli using intra-cavity generated non-circular beam of inhomogeneous intensity
R. Kumar, C. Shakher and D. S. Mehta
We report 3D multiple trapping of dielectric polystyrene (PS) beads and gold nano-particles (GNPs) in single beam optical tweezers system using an asymmetric beam of inhomogeneous intensity distribution. This special kind of beam of quasi-TEM11 profile was generated from intra-cavity CW-laser source operating at 532 nm. Multiple trapping of both the low refractive index rod-like Escherichia coli bacteria and 253 nm plasmonic GNPs dispersed in 1.025 μm PS beads which were homogenized in de-ionized water was realized utilizing this spatial beam. Laser-GNPs interaction rendered the enhancement of local surface plasmon resonance field around GNPs causing long-range aggregation of PS beads. The multiple trapping of plasmonic GNPs by the present simple method might find applications for micro- and nano-connectors, underlying physical processes in light-matter interaction assays for inter-particle force analysis, cancer diagnostic and photothermolysis, surface-enhanced Raman scattering (SERS) spectroscopy, and surface plasmon based biological and chemical sensors.
We report 3D multiple trapping of dielectric polystyrene (PS) beads and gold nano-particles (GNPs) in single beam optical tweezers system using an asymmetric beam of inhomogeneous intensity distribution. This special kind of beam of quasi-TEM11 profile was generated from intra-cavity CW-laser source operating at 532 nm. Multiple trapping of both the low refractive index rod-like Escherichia coli bacteria and 253 nm plasmonic GNPs dispersed in 1.025 μm PS beads which were homogenized in de-ionized water was realized utilizing this spatial beam. Laser-GNPs interaction rendered the enhancement of local surface plasmon resonance field around GNPs causing long-range aggregation of PS beads. The multiple trapping of plasmonic GNPs by the present simple method might find applications for micro- and nano-connectors, underlying physical processes in light-matter interaction assays for inter-particle force analysis, cancer diagnostic and photothermolysis, surface-enhanced Raman scattering (SERS) spectroscopy, and surface plasmon based biological and chemical sensors.
Dynamic deformation of red blood cell in Dual-trap Optical Tweezers
Sebastien Rancourt-Grenier, Ming-Tzo Wei, Jar-Jin Bai, Arthur Chiou, Paul P. Bareil, Pierre-Luc Duval, and Yunlong Sheng
Three-dimensional dynamic deformation of a red blood cell in a dual-trap optical tweezers is computed with the elastic membrane theory and is compared with the experimental results. When a soft particle is trapped by a laser beam, the particle is deformed depending on the radiation stress distribution whereas the stress distribution on the particle in turn depends on the deformation of its morphological shape. We compute the stress re-distribution on the deformed cell and its subsequent deformations recursively until a final equilibrium state solution is achieved. The experiment is done with the red blood cells in suspension swollen to spherical shape. The cell membrane elasticity coefficient is obtained by fitting the theoretical prediction with the experimental data. This approach allows us to evaluate up to 20% deformation of cell’s shape.
Three-dimensional dynamic deformation of a red blood cell in a dual-trap optical tweezers is computed with the elastic membrane theory and is compared with the experimental results. When a soft particle is trapped by a laser beam, the particle is deformed depending on the radiation stress distribution whereas the stress distribution on the particle in turn depends on the deformation of its morphological shape. We compute the stress re-distribution on the deformed cell and its subsequent deformations recursively until a final equilibrium state solution is achieved. The experiment is done with the red blood cells in suspension swollen to spherical shape. The cell membrane elasticity coefficient is obtained by fitting the theoretical prediction with the experimental data. This approach allows us to evaluate up to 20% deformation of cell’s shape.
Wednesday, May 5, 2010
Actin-like cytoskeleton filaments contribute to cell mechanics in bacteria
Siyuan Wang, Hugo Arellano-Santoyo, Peter A. Combs and Joshua W. Shaevitz
A filamentous cytoskeleton largely governs the physical shape and mechanical properties of eukaryotic cells. In bacteria, proteins homologous to all three classes of eukaryotic cytoskeletal filaments have recently been discovered. These proteins are essential for the maintenance of bacterial cell shape and have been shown to guide the localization of key cell-wall-modifying enzymes. However, whether the bacterial cytoskeleton is stiff enough to affect the overall mechanical rigidity of a cell has not been probed. Here, we used an optical trap to measure the bending rigidity of live Escherichia coli cells. We find that the actin-homolog MreB contributes nearly as much to the stiffness of a cell as the peptidoglycan cell wall. By quantitatively modeling these measurements, our data indicate that the MreB is rigidly linked to the cell wall, increasing the mechanical stiffness of the overall system. These data are the first evidence that the bacterial cytoskeleton contributes to the mechanical integrity of a cell in much the same way as it does in eukaryotes.
A filamentous cytoskeleton largely governs the physical shape and mechanical properties of eukaryotic cells. In bacteria, proteins homologous to all three classes of eukaryotic cytoskeletal filaments have recently been discovered. These proteins are essential for the maintenance of bacterial cell shape and have been shown to guide the localization of key cell-wall-modifying enzymes. However, whether the bacterial cytoskeleton is stiff enough to affect the overall mechanical rigidity of a cell has not been probed. Here, we used an optical trap to measure the bending rigidity of live Escherichia coli cells. We find that the actin-homolog MreB contributes nearly as much to the stiffness of a cell as the peptidoglycan cell wall. By quantitatively modeling these measurements, our data indicate that the MreB is rigidly linked to the cell wall, increasing the mechanical stiffness of the overall system. These data are the first evidence that the bacterial cytoskeleton contributes to the mechanical integrity of a cell in much the same way as it does in eukaryotes.
Direct Measurements of Heating by Electromagnetically Trapped Gold Nanoparticles on Supported Lipid Bilayers
Poul M. Bendix, S. Nader S. Reihani† and Lene B. Oddershede
Absorption of electromagnetic irradiation results in significant heating of metallic nanoparticles, an effect which can be advantageously used in biomedical contexts. Also, metallic nanoparticles are presently finding widespread use as handles, contacts, or markers in nanometer scale systems, and for these purposes it is essential that the temperature increase associated with electromagnetic irradiation is not harmful to the environment. Regardless of whether the heating of metallic nanoparticles is desired or not, it is crucial for nanobio assays to know the exact temperature increase associated with electromagnetic irradiation of metallic nanoparticles. We performed direct measurements of the temperature surrounding single gold nanoparticles optically trapped on a lipid bilayer, a biologically relevant matrix. The lipid bilayer had incorporated fluorescent molecules which have a preference for either fluid or gel phases. The heating associated with electromagnetic radiation was measured by visualizing the melted footprint around the irradiated particle. The effect was measured for individual gold nanoparticles of a variety of sizes and for a variety of laser powers. The temperatures were highly dependent on particle size and laser power, with surface temperature increments ranging from a few to hundreds of degrees Celsius. Our results show that by a careful choice of gold nanoparticle size and strength of irradiating electromagnetic field, one can control the exact particle temperature. The method is easily applicable to any type of nanoparticle for which the photothermal effect is sought to be quantified.
Absorption of electromagnetic irradiation results in significant heating of metallic nanoparticles, an effect which can be advantageously used in biomedical contexts. Also, metallic nanoparticles are presently finding widespread use as handles, contacts, or markers in nanometer scale systems, and for these purposes it is essential that the temperature increase associated with electromagnetic irradiation is not harmful to the environment. Regardless of whether the heating of metallic nanoparticles is desired or not, it is crucial for nanobio assays to know the exact temperature increase associated with electromagnetic irradiation of metallic nanoparticles. We performed direct measurements of the temperature surrounding single gold nanoparticles optically trapped on a lipid bilayer, a biologically relevant matrix. The lipid bilayer had incorporated fluorescent molecules which have a preference for either fluid or gel phases. The heating associated with electromagnetic radiation was measured by visualizing the melted footprint around the irradiated particle. The effect was measured for individual gold nanoparticles of a variety of sizes and for a variety of laser powers. The temperatures were highly dependent on particle size and laser power, with surface temperature increments ranging from a few to hundreds of degrees Celsius. Our results show that by a careful choice of gold nanoparticle size and strength of irradiating electromagnetic field, one can control the exact particle temperature. The method is easily applicable to any type of nanoparticle for which the photothermal effect is sought to be quantified.
Tuesday, May 4, 2010
Optimal beam diameter for optical tweezers
Akbar Samadi and Nader S. Reihani
An optimized optical trap is a favorable choice for nanoparticle trapping and micromanipulation of biological tissues. The collimation of laser beam before the objective can have significant influence on the trap by controlling the effective NA of the system. We have shown by both theory and experiment that in the aberration-free condition the filling factor of W∕D≃0.67 provides the strongest trap in the lateral directions for a micrometer-size bead. In this condition improvements up to ≃117%(≃168%), compared with the previously suggested ratio of W∕D≃1(W∕D≃1.25), were achieved in the lateral direction.
An optimized optical trap is a favorable choice for nanoparticle trapping and micromanipulation of biological tissues. The collimation of laser beam before the objective can have significant influence on the trap by controlling the effective NA of the system. We have shown by both theory and experiment that in the aberration-free condition the filling factor of W∕D≃0.67 provides the strongest trap in the lateral directions for a micrometer-size bead. In this condition improvements up to ≃117%(≃168%), compared with the previously suggested ratio of W∕D≃1(W∕D≃1.25), were achieved in the lateral direction.
Unfolding the A2 Domain of Von Willebrand Factor with the Optical Trap
Junyi Ying, Yingchen Ling, Lisa A. Westfield, J. Evan Sadler and Jin-Yu Shao
Von Willebrand factor (VWF) is a multimeric plasma glycoprotein involved in both hemostasis and thrombosis. VWF conformational changes, especially unfolding of the A2 domain, may be required for efficient enzymatic cleavage in vivo. It has been shown that a single A2 domain unfolds at most probable unfolding forces of 7–14 pN at force loading rates of 0.35–350 pN/s and A2 unfolding facilitates A2 cleavage in vitro. However, it remains unknown how much force is required to unfold the A2 domain in the context of a VWF multimer where A2 may be stabilized by other domains like A1 and A3. With the optical trap, we stretched VWF multimers and a poly-protein (A1A2A3)3 that contains three repeats of the triplet A1A2A3 domains at constant speeds of 2000 nm/s and 400 nm/s, respectively, which yielded corresponding average force loading rates of 90 and 22 pN/s. We found that VWF multimers became stiffer when they were stretched and extended by force. After force increased to a certain level, sudden extensional jumps that signify domain unfolding were often observed. Histograms of the unfolding force and the unfolded contour length showed two or three peaks that were integral multiples of 21 pN and 63 nm, respectively. Stretching of (A1A2A3)3 yielded comparable distributions of unfolding force and unfolded contour length, showing that unfolding of the A2 domain accounts for the behavior of VWF multimers under tension. These results show that the A2 domain can be indeed unfolded in the presence of A1, A3, and other domains. Compared with the value in the literature, the larger most probable unfolding force measured in this study suggests that the A2 domain is mechanically stabilized by A1 or A3 although variations in experimental setups and conditions may complicate this interpretation.
Use of optical tweezers to probe epithelial mechanosensation
Andrew Resnick
Cellular mechanosensation mechanisms have been implicated in a variety of diseasestates. Specifically in renal tubules, the primary cilium and associated mechanosensitive ion channels are hypothesized to play a role in water and salt homeostasis, with relevant disease states including polycystic kidney disease and hypertension. Previous experiments investigating ciliary-mediated cellular mechanosensation have used either fluid flow chambers or micropipetting to elicit a biological response. The interpretation of these experiments in terms of the “ciliary hypothesis” has been difficult due the spatiallydistributed nature of the mechanical disturbance—several competing hypotheses regarding possible roles of primary cilium, glycocalyx, microvilli, cell junctions, and actincytoskeleton exist. I report initial data using optical tweezers to manipulate individual primary cilia in an attempt to elicit a mechanotransduction response—specifically, the release of intracellular calcium. The advantage of using laser tweezers over previous work is that the applied disturbance is highly localized. I find that stimulation of a primary cilium elicits a response, while stimulation of the apical surface membrane does not. These results lend support to the hypothesis that the primary cilium mediates transduction of mechanical strain into a biochemical response in renal epithelia.
Cellular mechanosensation mechanisms have been implicated in a variety of diseasestates. Specifically in renal tubules, the primary cilium and associated mechanosensitive ion channels are hypothesized to play a role in water and salt homeostasis, with relevant disease states including polycystic kidney disease and hypertension. Previous experiments investigating ciliary-mediated cellular mechanosensation have used either fluid flow chambers or micropipetting to elicit a biological response. The interpretation of these experiments in terms of the “ciliary hypothesis” has been difficult due the spatiallydistributed nature of the mechanical disturbance—several competing hypotheses regarding possible roles of primary cilium, glycocalyx, microvilli, cell junctions, and actincytoskeleton exist. I report initial data using optical tweezers to manipulate individual primary cilia in an attempt to elicit a mechanotransduction response—specifically, the release of intracellular calcium. The advantage of using laser tweezers over previous work is that the applied disturbance is highly localized. I find that stimulation of a primary cilium elicits a response, while stimulation of the apical surface membrane does not. These results lend support to the hypothesis that the primary cilium mediates transduction of mechanical strain into a biochemical response in renal epithelia.
Monday, May 3, 2010
Optical Forces in Plasmonic Nanoparticle Dimers
Vladimir D. Miljkovi, Tavakol Pakizeh, Borja Sepulveda, Peter Johansson and Mikael Kall
We present calculations of the optical forces between two metal nanospheres forming a hybridized plasmonic dimer. We consider homo- and heterodimers and investigate different plane wave illumination configurations. The forces between the particles are calculated using full Mie theory combined with the Maxwell stress tensor (MST) formalism, as well as by approximate methods, such as the Lorentz force (LF) approach taken in the dipole limit and calculations based on an optical potential. We show that the simplified calculation schemes can lead to serious errors in the case of strongly interacting particles and low damping. In particular, we find that equilibrium configurations, corresponding to vanishing optical forces, only are possible for homodimers illuminated in the end-fire configuration and for heterodimers, although multipolar effects and damping radically reduce the repulsive interactions in the latter case.
We present calculations of the optical forces between two metal nanospheres forming a hybridized plasmonic dimer. We consider homo- and heterodimers and investigate different plane wave illumination configurations. The forces between the particles are calculated using full Mie theory combined with the Maxwell stress tensor (MST) formalism, as well as by approximate methods, such as the Lorentz force (LF) approach taken in the dipole limit and calculations based on an optical potential. We show that the simplified calculation schemes can lead to serious errors in the case of strongly interacting particles and low damping. In particular, we find that equilibrium configurations, corresponding to vanishing optical forces, only are possible for homodimers illuminated in the end-fire configuration and for heterodimers, although multipolar effects and damping radically reduce the repulsive interactions in the latter case.
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