Charles L. Asbury
The separation of sister chromatids during anaphase is the culmination of mitosis and one of the most strikingly beautiful examples of cellular movement. It consists of two distinct processes: Anaphase A, the movement of chromosomes toward spindle poles via shortening of the connecting fibers, and anaphase B, separation of the two poles from one another via spindle elongation. I focus here on anaphase A chromosome-to-pole movement. The chapter begins by summarizing classical observations of chromosome movements, which support the current understanding of anaphase mechanisms. Live cell fluorescence microscopy studies showed that poleward chromosome movement is associated with disassembly of the kinetochore-attached microtubule fibers that link chromosomes to poles. Microtubule-marking techniques established that kinetochore-fiber disassembly often occurs through loss of tubulin subunits from the kinetochore-attached plus ends. In addition, kinetochore-fiber disassembly in many cells occurs partly through ‘flux’, where the microtubules flow continuously toward the poles and tubulin subunits are lost from minus ends. Molecular mechanistic models for how load-bearing attachments are maintained to disassembling microtubule ends, and how the forces are generated to drive these disassembly-coupled movements, are discussed.
DOI
Friday, March 3, 2017
Radiation forces on a Rayleigh dielectric sphere produced by highly focused parabolic scaling Bessel beams
Mengwen Guo and Daomu Zhao
The radiation forces on a Rayleigh dielectric particle induced by a highly focused parabolic scaling Bessel beam (PSBB) are investigated. Numerical results show that the zero-order PSBB can be used to trap a high-index particle at the focus and near the focus by the first-order PSBB. For the low-index particle, it can be guided or confined in the dark core of the nonzero-order PSBB but cannot be stably trapped in this single-beam trap. Further, we analyze the condition of trapping stability. It is found that the lower limit in the particle radius for stable trapping is different for different orders.
DOI
The radiation forces on a Rayleigh dielectric particle induced by a highly focused parabolic scaling Bessel beam (PSBB) are investigated. Numerical results show that the zero-order PSBB can be used to trap a high-index particle at the focus and near the focus by the first-order PSBB. For the low-index particle, it can be guided or confined in the dark core of the nonzero-order PSBB but cannot be stably trapped in this single-beam trap. Further, we analyze the condition of trapping stability. It is found that the lower limit in the particle radius for stable trapping is different for different orders.
DOI
Direct measurement of cortical force generation and polarization in a living parasite
Rachel V. Stadler, Lauren A. White, Ke Hu, Brian P. Helmke, and William H. Guilford
Apicomplexa is a large phylum of intracellular parasites that are notable for the diseases they cause, including toxoplasmosis, malaria and cryptosporidiosis. A conserved motile system is critical to their lifecycles as it drives directional gliding motility between cells, as well as invasion of and egress from host cells. However, our understanding of this system is limited by a lack of measurements of the forces driving parasite motion. We used a laser trap to measure the function of the motility apparatus of living Toxoplasma gondii by adhering a microsphere to the surface of an immobilized parasite. Motion of the microsphere reflected underlying forces exerted by the motile apparatus. We found that force generated at the parasite surface begins with no preferential directionality, but becomes directed toward the rear of the cell after a period of time. The transition from non-directional to directional force generation occurs on spatial intervals consistent with the lateral periodicity of structures associated with the membrane pellicle, and is influenced by the kinetics of actin filament polymerization and cytoplasmic calcium. A lysine methyltransferase regulates both the magnitude and polarization of the force. Our work provides a novel means to dissect the motile mechanisms of these pathogens.
DOI
Apicomplexa is a large phylum of intracellular parasites that are notable for the diseases they cause, including toxoplasmosis, malaria and cryptosporidiosis. A conserved motile system is critical to their lifecycles as it drives directional gliding motility between cells, as well as invasion of and egress from host cells. However, our understanding of this system is limited by a lack of measurements of the forces driving parasite motion. We used a laser trap to measure the function of the motility apparatus of living Toxoplasma gondii by adhering a microsphere to the surface of an immobilized parasite. Motion of the microsphere reflected underlying forces exerted by the motile apparatus. We found that force generated at the parasite surface begins with no preferential directionality, but becomes directed toward the rear of the cell after a period of time. The transition from non-directional to directional force generation occurs on spatial intervals consistent with the lateral periodicity of structures associated with the membrane pellicle, and is influenced by the kinetics of actin filament polymerization and cytoplasmic calcium. A lysine methyltransferase regulates both the magnitude and polarization of the force. Our work provides a novel means to dissect the motile mechanisms of these pathogens.
DOI
Directed rotational motion of birefringent particles by randomly changing the barrier height at the threshold in a washboard potential
Basudev Roy, Erik Schaffer
This communication presents a simulation study of the directed rotational motion of a birefringent spherical particle trapped in optical tweezers with randomly varying ellipticity of a trapping light at the point of threshold. When noise is not applied, the potential barrier due to the linear component of the polarization is simulated to be sufficient to prevent directed rotation till a certain threshold value of ellipticity. Random variations to the ellipticity cause random variations in the barrier height, including instants when the barrier is lower than the threshold energy level. It is due to this that the particle exhibits directed rotational motion at ellipticity lower than the threshold value. We also examine the rotational velocity of the birefringent particle as a function of the extent of zero-mean random noise applied to the ellipticity.
DOI
This communication presents a simulation study of the directed rotational motion of a birefringent spherical particle trapped in optical tweezers with randomly varying ellipticity of a trapping light at the point of threshold. When noise is not applied, the potential barrier due to the linear component of the polarization is simulated to be sufficient to prevent directed rotation till a certain threshold value of ellipticity. Random variations to the ellipticity cause random variations in the barrier height, including instants when the barrier is lower than the threshold energy level. It is due to this that the particle exhibits directed rotational motion at ellipticity lower than the threshold value. We also examine the rotational velocity of the birefringent particle as a function of the extent of zero-mean random noise applied to the ellipticity.
DOI
Thursday, March 2, 2017
Atomization efficiency and photon yield in laser-induced breakdown spectroscopy analysis of single nanoparticles in an optical trap
Pablo Purohit, Francisco J. Fortes, J. Javier Laserna
Laser-induced breakdown spectroscopy (LIBS) was employed for investigating the influence of particle size on the dissociation efficiency and the absolute production of photons per mass unit of airborne solid graphite spheres under single-particle regime. Particles of average diameter of 400 nm were probed and compared with 2 μm particles. Samples were first catapulted into aerosol form and then secluded in an optical trap set by a 532 nm laser. Trap stability was quantified before subjecting particles to LIBS analysis. Fine alignment of the different lines comprising the optical catapulting-optical trapping-laser-induced breakdown spectroscopy instrument and tuning of excitation parameters conditioning the LIBS signal such as fluence and acquisition delay are described in detail with the ultimate goal of acquiring clear spectroscopic data on masses as low as 75 fg. The atomization efficiency and the photon yield increase as the particle size becomes smaller. Time-resolved plasma imaging studies were conducted to elucidate the mechanisms leading to particle disintegration and excitation.
DOI
Laser-induced breakdown spectroscopy (LIBS) was employed for investigating the influence of particle size on the dissociation efficiency and the absolute production of photons per mass unit of airborne solid graphite spheres under single-particle regime. Particles of average diameter of 400 nm were probed and compared with 2 μm particles. Samples were first catapulted into aerosol form and then secluded in an optical trap set by a 532 nm laser. Trap stability was quantified before subjecting particles to LIBS analysis. Fine alignment of the different lines comprising the optical catapulting-optical trapping-laser-induced breakdown spectroscopy instrument and tuning of excitation parameters conditioning the LIBS signal such as fluence and acquisition delay are described in detail with the ultimate goal of acquiring clear spectroscopic data on masses as low as 75 fg. The atomization efficiency and the photon yield increase as the particle size becomes smaller. Time-resolved plasma imaging studies were conducted to elucidate the mechanisms leading to particle disintegration and excitation.
DOI
Rich stochastic dynamics of co-doped Er:Yb fluorescence upconversion nanoparticles in the presence of thermal, non-conservative, harmonic and optical forces
Rene A Nome, Cecilia Sorbello, Matías Jobbágy, Beatriz C Barja, Vitor Sanches, Joyce S Cruz and Vinicius F Aguiar
The stochastic dynamics of individual co-doped Er:Yb upconversion nanoparticles (UCNP) were investigated from experiments and simulations. The UCNP were characterized by high-resolution scanning electron microscopy, dynamic light scattering, and zeta potential measurements. Single UCNP measurements were performed by fluorescence upconversion micro-spectroscopy and optical trapping. The mean-square displacement (MSD) from single UCNP exhibited a time-dependent diffusion coefficient which was compared with Brownian dynamics simulations of a viscoelastic model of harmonically bound spheres. Experimental time-dependent two-dimensional trajectories of individual UCNP revealed correlated two-dimensional nanoparticle motion. The measurements were compared with stochastic trajectories calculated in the presence of a non-conservative rotational force field. Overall, the complex interplay of UCNP adhesion, thermal fluctuations and optical forces led to a rich stochastic behavior of these nanoparticles.
DOI
The stochastic dynamics of individual co-doped Er:Yb upconversion nanoparticles (UCNP) were investigated from experiments and simulations. The UCNP were characterized by high-resolution scanning electron microscopy, dynamic light scattering, and zeta potential measurements. Single UCNP measurements were performed by fluorescence upconversion micro-spectroscopy and optical trapping. The mean-square displacement (MSD) from single UCNP exhibited a time-dependent diffusion coefficient which was compared with Brownian dynamics simulations of a viscoelastic model of harmonically bound spheres. Experimental time-dependent two-dimensional trajectories of individual UCNP revealed correlated two-dimensional nanoparticle motion. The measurements were compared with stochastic trajectories calculated in the presence of a non-conservative rotational force field. Overall, the complex interplay of UCNP adhesion, thermal fluctuations and optical forces led to a rich stochastic behavior of these nanoparticles.
DOI
Hot-nanoparticle-mediated fusion of selected cells
Azra Bahadori, Lene B. Oddershede, Poul M. Bendix
Complete fusion of two selected cells allows for the creation of novel hybrid cells with inherited genetic properties from both original cells. Alternatively, via fusion of a selected cell with a selected vesicle, chemicals or genes can be directly delivered into the cell of interest, to control cellular reactions or gene expression. Here, we demonstrate how to perform an optically controlled fusion of two selected cells or of one cell and one vesicle. Fusion is mediated by laser irradiating plasmonic gold nanoparticles optically trapped between two cells (or a vesicle and a cell) of interest. This hot-particle-mediated fusion causes total mixing of the two cytoplasms and the two cell membranes resulting in formation of a new hybrid cell with an intact cell membrane and enzymatic activity following fusion. Similarly, fusion between a vesicle and a cell results in delivery of the vesicle cargo to the cytoplasm, and after fusion, the cell shows signs of viability. The method is an implementation of targeted drug delivery at the single-cell level and has a great potential for cellular control and design.
DOI
Complete fusion of two selected cells allows for the creation of novel hybrid cells with inherited genetic properties from both original cells. Alternatively, via fusion of a selected cell with a selected vesicle, chemicals or genes can be directly delivered into the cell of interest, to control cellular reactions or gene expression. Here, we demonstrate how to perform an optically controlled fusion of two selected cells or of one cell and one vesicle. Fusion is mediated by laser irradiating plasmonic gold nanoparticles optically trapped between two cells (or a vesicle and a cell) of interest. This hot-particle-mediated fusion causes total mixing of the two cytoplasms and the two cell membranes resulting in formation of a new hybrid cell with an intact cell membrane and enzymatic activity following fusion. Similarly, fusion between a vesicle and a cell results in delivery of the vesicle cargo to the cytoplasm, and after fusion, the cell shows signs of viability. The method is an implementation of targeted drug delivery at the single-cell level and has a great potential for cellular control and design.
DOI
Stable optical trapping and sensitive characterization of nanostructures using standing-wave Raman tweezers
Mu-ying Wu, Dong-xiong Ling, Lin Ling, William Li & Yong-qing Li
Optical manipulation and label-free characterization of nanoscale structures open up new possibilities for assembly and control of nanodevices and biomolecules. Optical tweezers integrated with Raman spectroscopy allows analyzing a single trapped particle, but is generally less effective for individual nanoparticles. The main challenge is the weak gradient force on nanoparticles that is insufficient to overcome the destabilizing effect of scattering force and Brownian motion. Here, we present standing-wave Raman tweezers for stable trapping and sensitive characterization of single isolated nanostructures with a low laser power by combining a standing-wave optical trap with confocal Raman spectroscopy. This scheme has stronger intensity gradients and balanced scattering forces, and thus can be used to analyze many nanoparticles that cannot be measured with single-beam Raman tweezers, including individual single-walled carbon nanotubes (SWCNT), graphene flakes, biological particles, SERS-active metal nanoparticles, and high-refractive semiconductor nanoparticles. This would enable sorting and characterization of specific SWCNTs and other nanoparticles based on their increased Raman fingerprints.
DOI
Optical manipulation and label-free characterization of nanoscale structures open up new possibilities for assembly and control of nanodevices and biomolecules. Optical tweezers integrated with Raman spectroscopy allows analyzing a single trapped particle, but is generally less effective for individual nanoparticles. The main challenge is the weak gradient force on nanoparticles that is insufficient to overcome the destabilizing effect of scattering force and Brownian motion. Here, we present standing-wave Raman tweezers for stable trapping and sensitive characterization of single isolated nanostructures with a low laser power by combining a standing-wave optical trap with confocal Raman spectroscopy. This scheme has stronger intensity gradients and balanced scattering forces, and thus can be used to analyze many nanoparticles that cannot be measured with single-beam Raman tweezers, including individual single-walled carbon nanotubes (SWCNT), graphene flakes, biological particles, SERS-active metal nanoparticles, and high-refractive semiconductor nanoparticles. This would enable sorting and characterization of specific SWCNTs and other nanoparticles based on their increased Raman fingerprints.
DOI
Optical Trap Loading of Dielectric Microparticles In Air
Haesung Park, Thomas W. LeBrun
We demonstrate a method to trap a selected dielectric microparticle in air using radiation pressure from a single-beam gradient optical trap. Randomly scattered dielectric microparticles adhered to a glass substrate are momentarily detached using ultrasonic vibrations generated by a piezoelectric transducer (PZT). Then, the optical beam focused on a selected particle lifts it up to the optical trap while the vibrationally excited microparticles fall back to the substrate. A particle may be trapped at the nominal focus of the trapping beam or at a position above the focus (referred to here as the levitation position) where gravity provides the restoring force. After the measurement, the trapped particle can be placed at a desired position on the substrate in a controlled manner.
In this protocol, an experimental procedure for selective optical trap loading in air is outlined. First, the experimental setup is briefly introduced. Second, the design and fabrication of a PZT holder and a sample enclosure are illustrated in detail. The optical trap loading of a selected microparticle is then demonstrated with step-by-step instructions including sample preparation, launching into the trap, and use of electrostatic force to excite particle motion in the trap and measure charge. Finally, we present recorded particle trajectories of Brownian and ballistic motions of a trapped microparticle in air. These trajectories can be used to measure stiffness or to verify optical alignment through time domain and frequency domain analysis. Selective trap loading enables optical tweezers to track a particle and its changes over repeated trap loadings in a reversible manner, thereby enabling studies of particle-surface interaction.
DOI
We demonstrate a method to trap a selected dielectric microparticle in air using radiation pressure from a single-beam gradient optical trap. Randomly scattered dielectric microparticles adhered to a glass substrate are momentarily detached using ultrasonic vibrations generated by a piezoelectric transducer (PZT). Then, the optical beam focused on a selected particle lifts it up to the optical trap while the vibrationally excited microparticles fall back to the substrate. A particle may be trapped at the nominal focus of the trapping beam or at a position above the focus (referred to here as the levitation position) where gravity provides the restoring force. After the measurement, the trapped particle can be placed at a desired position on the substrate in a controlled manner.
In this protocol, an experimental procedure for selective optical trap loading in air is outlined. First, the experimental setup is briefly introduced. Second, the design and fabrication of a PZT holder and a sample enclosure are illustrated in detail. The optical trap loading of a selected microparticle is then demonstrated with step-by-step instructions including sample preparation, launching into the trap, and use of electrostatic force to excite particle motion in the trap and measure charge. Finally, we present recorded particle trajectories of Brownian and ballistic motions of a trapped microparticle in air. These trajectories can be used to measure stiffness or to verify optical alignment through time domain and frequency domain analysis. Selective trap loading enables optical tweezers to track a particle and its changes over repeated trap loadings in a reversible manner, thereby enabling studies of particle-surface interaction.
DOI
Local Arp2/3-dependent actin assembly modulates applied traction force during apCAM adhesion site maturation
Kenneth B. Buck, Andrew W. Schaefer, Vincent T. Schoonderwoert, Matthew S. Creamer, Eric R. Dufresne, and Paul Forscher
Homophilic binding of immunoglobulin superfamily molecules such as the Aplysia cell adhesion molecule (apCAM) leads to actin filament assembly near nascent adhesion sites. Such actin assembly can generate significant localized forces that have not been characterized in the larger context of axon growth and guidance. We used apCAM-coated bead substrates applied to the surface of neuronal growth cones to characterize the development of forces evoked by varying stiffness of mechanical restraint. Unrestrained bead propulsion matched or exceeded rates of retrograde network flow and was dependent on Arp2/3 complex activity. Analysis of growth cone forces applied to beads at low stiffness of restraint revealed switching between two states: frictional coupling to retrograde flow and Arp2/3-dependent propulsion. Stiff mechanical restraint led to formation of an extensive actin cup matching the geometric profile of the bead target and forward growth cone translocation; pharmacological inhibition of the Arp2/3 complex or Rac attenuated F-actin assembly near bead binding sites, decreased the efficacy of growth responses, and blocked accumulation of signaling molecules associated with nascent adhesions. These studies introduce a new model for regulation of traction force in which local actin assembly forces buffer nascent adhesion sites from the mechanical effects of retrograde flow.
DOI
Homophilic binding of immunoglobulin superfamily molecules such as the Aplysia cell adhesion molecule (apCAM) leads to actin filament assembly near nascent adhesion sites. Such actin assembly can generate significant localized forces that have not been characterized in the larger context of axon growth and guidance. We used apCAM-coated bead substrates applied to the surface of neuronal growth cones to characterize the development of forces evoked by varying stiffness of mechanical restraint. Unrestrained bead propulsion matched or exceeded rates of retrograde network flow and was dependent on Arp2/3 complex activity. Analysis of growth cone forces applied to beads at low stiffness of restraint revealed switching between two states: frictional coupling to retrograde flow and Arp2/3-dependent propulsion. Stiff mechanical restraint led to formation of an extensive actin cup matching the geometric profile of the bead target and forward growth cone translocation; pharmacological inhibition of the Arp2/3 complex or Rac attenuated F-actin assembly near bead binding sites, decreased the efficacy of growth responses, and blocked accumulation of signaling molecules associated with nascent adhesions. These studies introduce a new model for regulation of traction force in which local actin assembly forces buffer nascent adhesion sites from the mechanical effects of retrograde flow.
DOI
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