Monday, April 26, 2010

Calibration of optically trapped nanotools

D M Carberry, S H Simpson, J A Grieve, Y Wang, H Schäfer, M Steinhart, R Bowman, G M Gibson, M J Padgett, S Hanna and M J Miles

Holographically trapped nanotools can be used in a novel form of force microscopy. By measuring the displacement of the tool in the optical traps, the contact force experienced by the probe can be inferred. In the following paper we experimentally demonstrate the calibration of such a device and show that its behaviour is independent of small changes in the relative position of the optical traps. Furthermore, we explore more general aspects of the thermal motion of the tool.

Measurement of red blood cell mechanics during morphological changes

YongKeun Park, Catherine A. Best, Kamran Badizadegan, Ramachandra R. Dasari, Michael S. Felda, Tatiana Kuriabovad, Mark L. Henlee, Alex J. Levinef, and Gabriel Popescu

The human red blood cell (RBC) membrane, a fluid lipid bilayer tethered to an elastic 2D spectrin network, provides the principal control of the cell’s morphology and mechanics. These properties, in turn, influence the ability of RBCs to transport oxygen in circulation. Current mechanical measurements of RBCs rely on external loads. Here we apply a noncontact optical interferometric technique to quantify the thermal fluctuations of RBC membranes with 3 nm accuracy over a broad range of spatial and temporal frequencies. Combining this technique with a new mathematical model describing RBC membrane undulations, we measure the mechanical changes of RBCs as they undergo a transition from the normal discoid shape to the abnormal echinocyte and spherical shapes. These measurements indicate that, coincident with this morphological transition, there is a significant increase in the membrane’s shear, area, and bending moduli. This mechanical transition can alter cell circulation and impede oxygen delivery.

Thursday, April 22, 2010

Monte-Carlo simulation of effective stiffness of time-sharing optical tweezers

Ren, Y., Wu, J., Zhong, M., Li, Y.

The Brownian motion of a polystyrene bead trapped in a time-sharing optical tweezers (TSOT) is numerically simulated by adopting Monte-Carlo technique. By analyzing the Brownian motion signal, the effective stiffness of a TSOT is acquired at different switching frequencies. Simulation results confirm that for a specific laser power and duty ratio, the effective stiffness varies with the frequency at low frequency range, while at high frequency range it keeps constant. Our results reveal that the switching frequency can be used to control the stability of time-sharing optical tweezers in a range.

Control of myosin-I force sensing by alternative splicing

Joseph M. Laakso, John H. Lewis, Henry Shuman, and E. Michael Ostap

Myosin-Is are molecular motors that link cellular membranes to the actin cytoskeleton, where they play roles in mechano-signal transduction and membrane trafficking. Some myosin-Is are proposed to act as force sensors, dynamically modulating their motile properties in response to changes in tension. In this study, we examined force sensing by the widely expressed myosin-I isoform, myo1b, which is alternatively spliced in its light chain binding domain (LCBD), yielding proteins with lever arms of different lengths. We found the actin-detachment kinetics of the splice isoforms to be extraordinarily tension-sensitive, with the magnitude of tension sensitivity to be related to LCBD splicing. Thus, in addition to regulating step-size, motility rates, and myosin activation, the LCBD is a key regulator of force sensing. We also found that myo1b is substantially more tension-sensitive than other myosins with similar length lever arms, indicating that different myosins have different tension-sensitive transitions.

Monday, April 19, 2010

Developing a Stochastic Dynamic Programming Framework for Optical Tweezer-Based Automated Particle Transport Operations

Banerjee, A. G.; Pomerance, A.; Losert, W.; Gupta, S. K.;

Automated particle transport using optical tweezers requires the use of motion planning to move the particle while avoiding collisions with randomly moving obstacles. This paper describes a stochastic dynamic programming based motion planning framework developed by modifying the discrete version of an infinite-horizon partially observable Markov decision process algorithm. Sample trajectories generated by this algorithm are presented to highlight effectiveness in crowded scenes and flexibility. The algorithm is tested using silica beads in a holographic tweezer set-up and data obtained from the physical experiments are reported to validate various aspects of the planning simulation framework. This framework is then used to evaluate the performance of the algorithm under a variety of operating conditions.

Kinesin velocity increases with the number of motors pulling against viscoelastic drag

Jason Gagliano, Matthew Walb, Brian Blaker, Jed C. Macosko and George Holzwarth

Although the properties of single kinesin molecular motors are well understood, it is not clear whether multiple motors pulling a single vesicle in a cell cooperate or interfere with one another. To learn how small numbers of motors interact, microtubule gliding assays were carried out with full-length Drosophila kinesin in a novel motility medium containing xanthan, a stiff, water-soluble polysaccharide. At 2 mg/ml xanthan, the zero-shear viscosity of this medium is 1,000 times the viscosity of water, similar to cellular viscosity. To mimic the rheological drag force on the motors when attached to a vesicle in a cell, we attached a 2 μm bead to one end of the microtubule (MT). During gliding assays in our novel medium, the moving bead exerted a drag force of 4–15 pN on the kinesins pulling the MT. The velocity of MTs with an attached bead increased with MT length and with kinesin concentration. The increase with MT length arose because the number of motors is directly proportional to MT length. Our results show that small numbers of kinesins cooperate constructively when pulling against a viscoelastic drag. In the absence of a bead but still in the viscous medium, MT velocity was independent of MT length and kinesin concentration because the thin MT, like a snake moving through grass, was able to move between xanthan molecules with little resistance. A minimal shared-load model in which the number of motors is proportional to MT length fits the observed dependence of gliding velocity on MT length and kinesin concentration.

Longer axial trap distance and larger radial trap stiffness using a double-ring radially polarized beam

Yaoju Zhang, Taikei Suyama, and Biaofeng Ding

The optical trapping forces acting on a metallic Rayleigh particle are calculated for the case where a double-ring-shaped radially polarized beam is applied. The influence of the off-focus distance and the off-axis distance of a trapping particle on the trapping force is investigated. Compared with the use of the conventional single-ring-shaped radially polarized beam, the longer axial trap distance and the larger radial trap stiffness are predicted using a double-ring-shaped radially polarized beam in an optical trap. These features are useful for improving the trapping ability of an optical trap system where a longer axial trap distance is needed.

Multiscale simulation of erythrocyte membranes

Zhangli Peng, Robert J. Asaro, and Qiang Zhu

To quantitatively predict the mechanical response and mechanically induced remodeling of red blood cells, we developed a multiscale method to correlate distributions of internal stress with overall cell deformation. This method consists of three models at different length scales: in the complete cell level the membrane is modeled as two distinct layers of continuum shells using finite element method (Level III), in which the skeleton-bilayer interactions are depicted as a slide in the lateral (i.e., in-plane) direction (caused by the mobility of the skeleton-bilayer pinning points) and a normal contact force; the constitutive laws of the inner layer (the protein skeleton) are obtained from a molecular-based model (Level II); the mechanical properties of the spectrin (Sp, a key component of the skeleton), including its folding/unfolding reactions, are obtained with a stress-strain model (Level I). Model verification is achieved through comparisons with existing numerical and experimental studies in terms of the resting shape of the cell as well as cell deformations induced by micropipettes and optical tweezers. Detailed distributions of the interaction force between the lipid bilayer and the skeleton that may cause their dissociation and lead to phenomena such as vesiculation are predicted. Specifically, our model predicts correlation between the occurrence of Sp unfolding and increase in the mechanical load upon individual skeleton-bilayer pinning points. Finally a simulation of the necking process after skeleton-bilayer dissociation, a precursor of vesiculation, is conducted.

Voltage-induced bending and electromechanical coupling in lipid bilayers

Ben Harland, William E. Brownell, Alexander A. Spector, and Sean X. Sun

The electrical properties of the cellular membrane are important for ion transport across cells and electrophysiology. Plasma membranes also resist bending and stretching, and mechanical properties of the membrane influence cell shape and forces in membrane tethers pulled from cells. There exists a coupling between the electrical and mechanical properties of the membrane. Previous work has shown that applied voltages can induce forces and movements in the lipid bilayer. We present a theory that computes membrane bending deformations and forces as the applied voltage is changed. We discover that electromechanical coupling in lipid bilayers depends on the voltage-dependent adsorption of ions into the region occupied by the phospholipid head groups. A simple model of counter-ion absorption is investigated. We show that electromechanical coupling can be measured using membrane tethers and we use our model to predict the membrane tether tension as a function of applied voltage. We also discuss how electromechanical coupling in membranes may influence transmembrane protein function.

Hydrodynamic synchronization of colloidal oscillators

Jurij Kotar, Marco Leoni, Bruno Bassetti, Marco Cosentino Lagomarsino, and Pietro Cicuta

Two colloidal spheres are maintained in oscillation by switching the position of an optical trap when a sphere reaches a limit position, leading to oscillations that are bounded in amplitude but free in phase and period. The interaction between the oscillators is only through the hydrodynamic flow induced by their motion. We prove that in the absence of stochastic noise the antiphase dynamical state is stable, and we show how the period depends on coupling strength. Both features are observed experimentally. As the natural frequencies of the oscillators are made progressively different, the coordination is quickly lost. These results help one to understand the origin of hydrodynamic synchronization and how the dynamics can be tuned. Cilia and flagella are biological systems coupled hydrodynamically, exhibiting dramatic collective motions. We propose that weakly correlated phase fluctuations, with one of the oscillators typically precessing the other, are characteristic of hydrodynamically coupled systems in the presence of thermal noise.

Contribution of the myosin VI tail domain to processive stepping and intramolecular tension sensing

Alexander R. Dunna, Peiying Chuana, Zev Bryantb, and James A. Spudich

Myosin VI is proposed to act as both a molecular transporter and as an anchor in vivo. A portion of the molecule C-terminal to the canonical lever arm, termed the medial tail (MT), has been proposed to act as either a lever arm extension or as a dimerization motif. We describe constructs in which the MT is interrupted by a glycine-rich molecular swivel. Disruption of the MT results in decreased processive run lengths measured using single-molecule fluorescence microscopy and a decreased step size under applied load as measured in an optical trap. We used single-molecule gold nanoparticle tracking and optical trapping to examine the mechanism of coordination between the heads of dimeric myosin VI. We detect two rate-limiting kinetic processes at low ATP concentrations. Our data can be explained by a model in which intramolecular tension greatly increases the affinity of the lead head for ADP, likely by slowing ADP release from the lead head. This mechanism likely increases both the motor's processivity and its ability to act as an anchor under physiological conditions.

Dark focal spot shaping of hyperbolic-cosine-Gaussian beam

Xiumin Gao, Qiufang Zhan, Jinsong Li, Song Hu, Jian Wang, and Songlin Zhuang

Dark focal spot shaping is investigated by vector diffraction theory in the focal region of a hyperbolic-cosine beam that contains one on-axis spiral optical vortex. Results show that a dark focal shape can be altered considerably by the decentered parameters in cosh parts of the beam and topological charge of the vortex. Many novel and interesting dark focal shapes may appear, including rhombic, quadrangular, cross-shaped, and foursquare dark foci. Some dark focal spot chains can also occur. In addition, the numerical aperture of the focusing system can also affect dark focal shapes remarkably, which may lead to dark focal spots that disappear or change focal shape due to the depolarization effect for a high numerical aperture. All of the above dark focal shapes can be used in an optical manipulation system to construct alterable optical traps.

On the Positioning Problem of a Microscopic Particle Trapped in Optical Tweezers

Carlos Aguilar-Ibañez and Luis I. Rosas-Soriano

We solve the positioning problem of a spherical microparticle trapped by Optical Tweezers, under the assumption that the drag viscous force is presented. To do it, we develop two control strategies for the manipulation of this kind of optical trap. The first control strategy is developed assuming that the damping coefficient is known, while in the second strategy this parameter value is only partially known, which in practice it is more realistic due to the difficulty to estimate it. Both strategies are based on the traditional Lyapunov method in conjunction with the use of a saturation function. The stability analysis of both strategies was carried out by using the standard Lyapunov stability theory. Finally, numerical simulations validate the effectiveness of both control approaches in reducing the random position fluctuations produced by the inherent thermal noise.


Thursday, April 8, 2010

Raman tweezers and their application to the study of singly trapped eukaryotic cells

Richard D. Snook, Timothy J. Harvey, Elsa Correia Faria and Peter Gardner

In this review the recent emergence of Raman tweezers as an analytical technique for single eukaryotic cell analysis is described. The Raman tweezer technique combines Raman spectroscopy as a diagnostic tool with optical tweezers by which means single cells can be trapped and manipulated in a laser beam using a high numerical aperture imaging microscope. Necessary instrumental requirements to facilitate Raman tweezer experiments are discussed together with practical considerations such as the potential for photodamage of cells subjected to trapping and Raman excitation. Specific applications of Raman tweezers to the analysis of cancer cells, erythrocytes and lymphocytes, micro-organisms and sub-cellular components e.g. chromosomes and mitochondria are then discussed followed by a summary of the future potential of the technique for single cell analysis.


Factors Affecting Daughter Cells' Arrangement during the Early Bacterial Divisions

Pin-Tzu Su, Pei-Wen Yen, Shao-Hung Wang, Chi-Hung Lin, Arthur Chiou, Wan-Jr Syu

On agar plates, daughter cells of Escherichia coli mutually slide and align side-by-side in parallel during the first round of binary fission. This phenomenon has been previously attributed to an elastic material that restricts apparently separated bacteria from being in string. We hypothesize that the interaction between bacteria and the underneath substratum may affect the arrangement of the daughter bacteria. To test this hypothesis, bacterial division on hyaluronic acid (HA) gel, as an alternative substratum, was examined. Consistent with our proposition, the HA gel differs from agar by suppressing the typical side-by-side alignments to a rare population. Examination of bacterial surface molecules that may contribute to the daughter cells' arrangement yielded an observation that, with disrupted lpp, the E. coli daughter cells increasingly formed non-typical patterns, i.e. neither sliding side-by-side in parallel nor forming elongated strings. Therefore, our results suggest strongly that the early cell patterning is affected by multiple interaction factors. With oscillatory optical tweezers, we further demonstrated that the interaction force decreased in bacteria without Lpp, a result substantiating our notion that the side-by-side sliding phenomenon directly reflects the strength of in-situ interaction between bacteria and substratum.

Wednesday, April 7, 2010

Optical sorting using an array of optical vortices with fractional topological charge

Cheng-Shan Guo, Ya-Nan Yu and Zhengping Hong

An array of optical vortices with fractional topological charge is generated using a phase-only Talbot array illuminator and used to sort microparticles. Our theoretical analysis and experimental results reveal that when a particle passes through a fractional vortex array, it will be driven by two forces, intensity-gradient force and phase-gradient force, and the cooperation of these two forces can improve its ability in optical sorting because of the special intensity and phase distribution of the fractional optical vortex array. Larger angle separation could be obtained with moderate laser power.

Thursday, April 1, 2010

Force Generation in Lamellipodia Is a Probabilistic Process with Fast Growth and Retraction Events

Rajesh Shahapure, Francesco Difato, Alessandro Laio, Giacomo Bisson, Erika Ercolini, Ladan Amin, Enrico Ferrari and Vincent Torre

Polymerization of actin filaments is the primary source of motility in lamellipodia and it is controlled by a variety of regulatory proteins. The underlying molecular mechanisms are only partially understood and a precise determination of dynamical properties of force generation is necessary. Using optical tweezers, we have measured with millisecond (ms) temporal resolution and picoNewton (pN) sensitivity the force-velocity (Fv) relationship and the power dissipated by lamellipodia of dorsal root ganglia neurons. When force and velocity are averaged over 3–5 s, the Fv relationships can be flat. On a finer timescale, random occurrence of fast growth and subsecond retractions become predominant. The maximal power dissipated by lamellipodia over a silica bead with a diameter of 1 μm is 10−16 W. Our results clarify the dynamical properties of force generation: i), force generation is a probabilistic process; ii), underlying biological events have a bandwidth up to at least 10 Hz; and iii), fast growth of lamellipodia leading edge alternates with local retractions.

Optical Trapping of Amino Acids in Aqueous Solutions

Yasuyuki Tsuboi, Tatsuya Shoji and Noboru Kitamura

In this paper, we demonstrated that the photon forces that are generated by a near-infrared (1064 nm) focused laser beam can manipulate (optically trap) small amino acid molecules in aqueous solutions. Under observation with an optical microscope, we observed the gradual growth of a particle-like assembly of objects at the focal point during laser irradiation into an aqueous arginine solution. The observation was a molecular assembly of arginine by means of confocal Raman microspectroscopy. Such molecular assemblies were also observed for other amino acids (glycine, proline, serine, and alanine), showing that the optical manipulation technique can be extensively applied to the micromanipulation of amino acids. From experimental observations and numeral calculations, we consider that the origin of the present manipulation phenomenon is ascribed to optical trapping, not of individual molecules, but of molecular clusters of amino acids.