Tuesday, August 31, 2010

Short-Range Force Detection Using Optically Cooled Levitated Microspheres

Andrew A. Geraci, Scott B. Papp, and John Kitching

We propose an experiment using optically trapped and cooled dielectric micro-spheres for the detection of short-range forces. The center-of-mass motion of a microsphere trapped in vacuum can experience extremely low dissipation and quality factors of 1012, leading to yoctonewton force sensitivity. Trapping the sphere in an optical field enables positioning at less than 1  μm from a surface, a regime where exotic new forces may exist. We expect that the proposed system could advance the search for non-Newtonian gravity forces via an enhanced sensitivity of 105–107 over current experiments at the 1  μm length scale. Moreover, our system may be useful for characterizing other short-range physics such as Casimir forces.


Laser trapping and picosecond time-resolved spectroscopy of water droplets in air: cavity-enhanced spontaneous emission of Ru(bpy)3Cl2

Shoji Ishizaka, Yuya Suzuki and Noboru Kitamura

Whispering gallery mode (WGM) resonances were observed in the emission spectrum of Ru(bpy)32+ (bpy = 2,2′-bipyridine) in a single laser-trapped water droplet levitated in air. The emission decay profiles of Ru(bpy)32+ in the water droplets comprised fast and slow decay components. The emission lifetime of the slow decay component was independent of the diameter of the droplet, and corresponded to the value in a bulk aqueous solution. On the other hand, the emission lifetime of the fast decay component decreased with decreasing the droplet diameter, which could be ascribed to the cavity-enhanced spontaneous emission. The decrease in the emission lifetime of the fast decay component as a function of the droplet diameter was explained on the basis of cavity quantum electrodynamic (QED) effects. It was shown that the mode characteristic of WGM resonances and the enhancement factor of the radiative rate of Ru(bpy)32+ were controlled by the size of the water droplet.


Light-Induced Agglomeration and Diffusion of Different Particles with Optical Tweezers

LI Xue-Cong, SUN Xiu-Dong, LIU Hong-Peng, ZHANG Jian-Long

The dynamic process of light-induced agglomeration of carbon nanotubes (CNTs), C60 and Escherichia coli (E.coli) in aqueous solutions is demonstrated using an optical tweezers system. Based on the results, the diameter of the agglomerated region and the agglomeration rate increase with the increasing laser power. After the saturationstable period, CNTs diffuse completely, C60 clusters only diffuse partially, and E. coli never diffuses in the agglomeration region. Theoretical analyses show that the molecular polarization and thermal diffusion of particles play crucial roles in the diffusion process. The results indicate the possibility of using light to aggregate and sort nanoparticles.


Monday, August 30, 2010

Dynamic and Reversible Organization of Zeolite L Crystals Induced by Holographic Optical Tweezers

Mike Woerdemann, Stefan Gläsener, Florian Hörner, André Devaux, Luisa De Cola, Cornelia Denz

Organization and patterning of zeolite L crystals with their unique properties such as their one-dimensional nano channel system is of highest topical interest with various applications in many areas of science. We demonstrate full three-dimensional optical control of single zeolite L crystals and for the first time fully reversible, dynamic organization of a multitude of individually controlled zeolite L crystals.


Passive torque wrench and angular position detection using a single-beam optical trap

James Inman, Scott Forth, and Michelle D. Wang

The recent advent of angular optical trapping techniques has allowed for rotational control and direct torque measurement on biological substrates. Here we present a method that increases the versatility and flexibility of these techniques. We demonstrate that a single beam with a rapidly rotating linear polarization can be utilized to apply a constant controllable torque to a trapped particle without active feedback, while simultaneously measuring the particle angular position. In addition, this device can rapidly switch between a torque wrench and an angular trap. These features should make possible torsional measurements across a wide range of biological systems.


Structured Post-IQ Domain Governs Selectivity of Myosin X for Fascin-Actin Bundles

Stanislav Nagy and Ronald S. Rock

Without guidance cues, cytoskeletal motors would traffic components to the wrong destination with disastrous consequences for the cell. Recently, we identified a motor protein, myosin X, that identifies bundled actin filaments for transport. These bundles direct myosin X to a unique destination, the tips of cellular filopodia. Because the structural and kinetic features that drive bundle selection are unknown, we employed a domain-swapping approach with the nonselective myosin V to identify the selectivity module of myosin X. We found a surprising role of the myosin X tail region (post-IQ) in supporting long runs on bundles. Moreover, the myosin X head is adapted for initiating processive runs on bundles. We found that the tail is structured and biases the orientation of the two myosin X heads because a targeted insertion that introduces flexibility in the tail abolishes selectivity. Together, these results suggest how myosin motors may manage to read cellular addresses.

Use of tapered amplifier diode laser for biological-friendly high-resolution optical trapping

Wei Cheng, Ximiao Hou, and Fangmao Ye

A 1064nm laser is commonly used for biological optical trapping. However, it has the problem of generating reactive oxygen species in the presence of a sensitizer, which leads to photo damage in biological samples. Here we constructed optical tweezers using a tapered amplifier diode laser that operates at 830nm. Compared to a 1064nm laser, this laser is friendly to live cells, eliminates photo damage associated with reactive oxygen species, and allows simultaneous two-photon fluorescence imaging of green fluorescent proteins in live mammalian cells. All these advantages could significantly benefit future application of this single molecule technique in biological studies.


Dependence of interparticle force on temperature and cell thickness in nematic colloids

Noboru Kondo, Yasutaka Iwashita, and Yasuyuki Kimura

We have experimentally studied the interparticle force between two particles accompanied by hyperbolic hedgehog defects in a nematic liquid crystal. The force F was measured with dual-beam optical tweezers at various temperatures and in cells with various thicknesses. In a thick cell, the dependence of F on the interparticle distance R obtained at different temperatures can be scaled to a universal curve of F∝R−4 for R>3a, where a is the radius of a particle. The effective elastic constant evaluated from F is found to be in good agreement with splay constant of the nematic liquid crystal. In a thin cell, the magnitude of F decreases and the dependence ofF on R becomes short-ranged as the thickness of a cell, L, decreases. The reduced force curves, FL4 against R/L, at different L are found to be scaled to a single theoretical curve which has been proposed recently.


Friday, August 27, 2010

Mechanical Characterization of One-Headed Myosin-V Using Optical Tweezers

Tomonobu M. Watanabe, Atsuko H. Iwane, Hiroto Tanaka, Mitsuo Ikebe, Toshio Yanagida

Class V myosin (myosin-V) is a cargo transporter that moves along an actin filament with large (~36-nm) successive steps. It consists of two heads that each includes a motor domain and a long (23 nm) neck domain. One of the more popular models describing these steps, the hand-over-hand model, assumes the two-headed structure is imperative. However, we previously succeeded in observing successive large steps by one-headed myosin-V upon optimizing the angle of the acto-myosin interaction. In addition, it was reported that wild type myosin-VI and myosin-IX, both one-headed myosins, can also generate successive large steps. Here, we describe the mechanical properties (stepsize and stepping kinetics) of successive large steps by one-headed and two-headed myosin-Vs. This study shows that the stepsize and stepping kinetics of one-headed myosin-V are very similar to those of the two-headed one. However, there was a difference with regards to stability against load and the number of multisteps. One-headed myosin-V also showed unidirectional movement that like two-headed myosin-V required 3.5 kBT from ATP hydrolysis. This value is also similar to that of smooth muscle myosin-II, a non-processive motor, suggesting the myosin family uses a common mechanism for stepping regardless of the steps being processive or non-processive. In this present paper, we conclude that one-headed myosin-V can produce successive large steps without following the hand-over-hand mechanism.


Local mechanical properties of a hyperswollen lyotropic lamellar phase

Naoki Yamamoto, Masatoshi Ichikawa, and Yasuyuki Kimura

The local and slow dynamics of a colloidal particle dispersed in a hyperswollen lyotropic lamellar phase was studied by passive and active microrheological methods. According to observation by a particle-tracking video microscopy, the motion of a particle follows a normal diffusion process in a lamellar phase with a large interlayer distance. However, trap-diffusion motion was also observed for a small interlayer distance. This characteristic motion was discussed by consideration of the time evolution of mean square displacement, the van Hove self-correlation function and the non-Gaussian parameter. These indicate that the dynamics of a particle becomes spatially heterogeneous in a lamellar phase whose interlayer distance is close to the size of the particle. By the measurement of local viscosity using optical tweezers, Newtonian behavior was observed at a high shear rate. In a lamellar phase with a small interlayer distance, non-Newtonian behavior was also observed at a low shear rate. The dependences of effective viscosity on the interlayer distance obtained by both methods are in agreement. The effective viscosity in the lamellar phase was found to be four to five times larger than that of water even for a large interlayer distance. Possible reasons for the increase in effective viscosity are discussed quantitatively.


Cell Membrane Tethers Generate Mechanical Force in Response to Electrical Stimulation

William E. Brownell, Feng Qian and Bahman Anvari

Living cells maintain a huge transmembrane electric field across their membranes. This electric field exerts a force on the membrane because the membrane surfaces are highly charged. We have measured electromechanical force generation by cell membranes using optically trapped beads to detach the plasma membrane from the cytoskeleton and form long thin cylinders (tethers). Hyperpolarizing potentials increased and depolarizing potentials decreased the force required to pull a tether. The membrane tether force in response to sinusoidal voltage signals was a function of holding potential, tether diameter, and tether length. Membrane electromechanical force production can occur at speeds exceeding those of ATP-based protein motors. By harnessing the energy in the transmembrane electric field, cell membranes may contribute to processes as diverse as outer hair cell electromotility, ion channel gating, and transport.


Thursday, August 26, 2010

Actin Cross-link Assembly and Disassembly Mechanics for {alpha}-Actinin and Fascin

Courson DS, Rock RS

Self-assembly of complex structures is commonplace in biology but often poorly understood. In the case of the actin cytoskeleton, a great deal is known about the components that include higher order structures, such as lamellar meshes, filopodial bundles, and stress fibers. Each of these cytoskeletal structures contains actin filaments and cross-linking proteins, but the role of cross-linking proteins in the initial steps of structure formation has not been clearly elucidated. We employ an optical trapping assay to investigate the behaviors of two actin cross-linking proteins, fascin and alpha-actinin, during the first steps of structure assembly. Here, we show that these proteins have distinct binding characteristics that cause them to recognize and cross-link filaments that are arranged with specific geometries. alpha-Actinin is a promiscuous cross-linker, linking filaments over all angles. It retains this flexibility after cross-links are formed, maintaining a connection even when the link is rotated. Conversely, fascin is extremely selective, only cross-linking filaments in a parallel orientation. Surprisingly, bundles formed by either protein are extremely stable, persisting for over 0.5 h in a continuous wash. However, using fluorescence recovery after photobleaching and fluorescence decay experiments, we find that the stable fascin population can be rapidly competed away by free fascin. We present a simple avidity model for this cross-link dissociation behavior. Together, these results place constraints on how cytoskeletal structures assemble, organize, and disassemble in vivo.


Unconventional Processive Mechanics of Non-muscle Myosin IIB

Melanie F. Norstrom, Philip A. Smithback and Ronald S. Rock

Proper tension maintenance in the cytoskeleton is essential for regulated cell polarity, cell motility, and division. Non-muscle myosin IIB (NMIIB) generates tension along actin filaments in many cell types, including neuronal, cardiac, and smooth muscle cells. Using a three-bead optical trapping assay, we recorded NMIIB interactions with actin filaments to determine if a NMIIB dimer cycles along an actin filament in a processive manner. Our results show that NMIIB is the first myosin II to exhibit evidence of processive stepping behavior. Analysis of these data reveals a forward displacement of 5.4 nm and, surprisingly, frequent backward steps of −5.9 nm. Processive stepping along the long pitch helix of actin may provide a mechanism for disassembly of fascin-actin bundles. Forward steps and detachment are weakly force-dependent at all forces, consistent with rate-limiting and force-dependent ADP release. However, backward steps are nearly force-independent. Our data support a model in which NMIIB can readily move in both directions at stall, which may be important for a general regulator of cytoskeleton tension.

Insight into helicase mechanism and function revealed through single-molecule approaches

Jaya G. Yodh, Michael Schlierf and Taekjip Ha

Helicases are a class of nucleic acid (NA) motors that catalyze NTP-dependent unwinding of NA duplexes into single strands, a reaction essential to all areas of NA metabolism. In the last decade, single-molecule (sm) technology has proven to be highly useful in revealing mechanistic insight into helicase activity that is not always detectable via ensemble assays. A combination of methods based on fluorescence, optical and magnetic tweezers, and flow-induced DNA stretching has enabled the study of helicase conformational dynamics, force generation, step size, pausing, reversal and repetitive behaviors during translocation and unwinding by helicases working alone and as part of multiprotein complexes. The contributions of these sm investigations to our understanding of helicase mechanism and function will be discussed.


Haptic Feedback of Piconewton Interactions with Optical Tweezers

Cécile Pacoret, Arvid Bergander and Stéphane Régnier

Haptic feedback for micro- and nanomanipulation is a research area of growing importance with many potential applications in micro- and biotechnology. Past research often involves the coupling of atomic force microscopes to haptic devices, but the results are not satisfactory. We propose to adopt a different approach, which consists of contactless manipulation, in particular by using optical tweezers, coupled with a haptic feedback device. In this article, we describe the potential of such a tool and show with some first experiments of stable interactions between micro-particles.


Friday, August 20, 2010

Confocal Raman Microscopy of Optical-Trapped Particles in Liquids

Daniel P. Cherney and Joel M. Harris

The in situ analysis of small, dispersed particles in liquids is a challenging problem, the successful solution to which influences diverse applications of colloidal particles in materials science, synthetic chemistry, and molecular biology. Optical trapping of small particles with a tightly focused laser beam can be combined with confocal Raman microscopy to provide molecular structure information about individual, femtogram-sized particles in liquid samples. In this review, we consider the basic principles of combining optical trapping and confocal Raman spectroscopy, then survey the applications that have been developed through the combination of these techniques and their use in the analysis of particles dispersed in liquids.


Light forces the pace: optical manipulation for biophotonics

David James Stevenson, Frank Gunn-Moore, Kishan Dholakia

The biomedical sciences have benefited immensely from photonics technologies in thelast 50 years. This includes the application of minute forces that enable the trapping and manipulation of cells and single molecules. In terms of the area of biophotonics, optical manipulation has made a seminal contribution to our understanding of the dynamics of single molecules and the microrheology of cells. Here we present a review of optical manipulation, emphasizing its impact on the areas of single-molecule studies and single-cell biology, and indicating some of the key experiments in the fields.


Tuesday, August 17, 2010

Nonlinear Elasticity and an 8-nm Working Stroke of Single Myosin Molecules in Myofilaments

Motoshi Kaya and Hideo Higuchi

Using optical trapping and fluorescence imaging techniques, we measured the step size and stiffness of single skeletal myosins interacting with actin filaments and arranged on myosin-rodcofilaments that approximate myosin mechanics during muscle contraction. Stiffness is dramatically lower for negatively compared to positively strained myosins, consistent with buckling of myosin’s subfragment 2 rod domain. Low stiffness minimizes drag of negatively strained myosins during contraction at loaded conditions. Myosin's elastic portion is stretched during active force generation, reducing apparent step size with increasing load, even though the working stroke is approximately constant at about 8 nanometers. Taking account of the nonlinear nature of myosin elasticity is essential to relate myosin’s internal structural changes to physiological force generation and filament sliding.


Monday, August 16, 2010

Flow-assisted Single-beam Optothermal Manipulation of Microparticles

Yangyang Liu and Andrew W. Poon

An optothermal tweezer was developed with a single-beam laser at 1550 nm for manipulation of colloidal microparticles. Strong absorption in water can thermally induce a localized flow, which exerts a Stokes’ drag on the particles that complements the gradient force. Long-range capturing of 6 μm polystyrene particles over ∼176 μm was observed with a tweezing power of ∼7 mW. Transportation and levitation, targeted deposition and selective levitation of particles were explored to experimentally demonstrate the versatility of the optothermal tweezer as a multipurpose particle manipulation tool.


Holographic optical manipulation of motor-driven membranous structures in living NG-108 cells

Arnau Farré, Carol López-Quesada, Jordi Andilla, Estela Martín-Badosa, and Mario Montes-Usategui

Optical tweezer experiments have partially unveiled the mechanical properties of processive motor proteins while driving polystyrene or silica microbeads in vitro. However, the set of forces underlying the more complex transport mechanisms in living samples remains poorly understood. Several studies have shown that optical tweezers are capable of trapping vesicles and organelles in the cytoplasm of living cells, which can be used as handles to mechanically interact with engaged (active) motors, or other components regulating transport. This may ultimately enable the exploration of the mechanics of this trafficking mechanism in vivo. These cell manipulation experiments have been carried out using different strategies to achieve dynamic beam steering capable of trapping thesesubcellular structures. We report here the first trapping and manipulation, to our knowledge, of such small motor-propelled cargos in living cells using holographic technology.


Optical manipulators of microparticles using femtosecond laser radiation

V V Buchanov, V A Derzhavin, A D Zalesskii, I V Reshetov, Oleg M Sarkisov and A I Shushin

Two setups for manipulating (translocation, stretching, rotation) microscopic objects (with the dimensions from several nanometers to tens of micrometers) are developed based on optical trapping by femtosecond laser radiation. The possibility of single-cell translocation is shown. The possibility of destructing malignant cells as well as of cutting off a fragment from a malignant cell cluster due to the rapture of bonds is demonstrated in multiphoton absorption of femtosecond light pulses. The possibility of the holographic control to move several particles simultaneously is shown.


Thursday, August 12, 2010

Doppler Cooling a Microsphere

P. F. Barker 

Doppler cooling the center-of-mass motion of an optically levitated microsphere via the velocity-dependent scattering force from narrow whispering gallery mode resonances is described. Light that is red detuned from the whispering gallery mode resonance can be used to damp the center-of-mass motion in a process analogous to the Doppler cooling of atoms. The scattering force is not limited by saturation but can be controlled by the incident power. Cooling times on the order of seconds are calculated for a 20 μm diameter silica microsphere trapped within optical tweezers.


Force-dependent polymorphism in type IV pili reveals hidden epitopes

Nicolas Biais, Dustin L. Higashi, Jasna Brujić, Magdalene So, and Michael P. Sheetz

Through evolution, nature has produced exquisite nanometric structures, with features unrealized in the most advanced man-made devices. Type IV pili (Tfp) represent such a structure: 6-nm-wide retractable filamentous appendages found in many bacteria, including human pathogens. Whereas the structure of Neisseria gonorrhoeae Tfp has been defined by conventional structural techniques, it remains difficult to explain the wide spectrum of functions associated with Tfp. Here we uncover a previously undescribed force-induced quaternary structure of the N. gonorrhoeae Tfp. By using a combination of optical and magnetic tweezers, atomic force microscopy, and molecular combing to apply forces on purified Tfp, we demonstrate that Tfp subjected to approximately 100 pN of force will transition into a new conformation. The new structure is roughly 3 times longer and 40% narrower than the original structure. Upon release of the force, the Tfp fiber regains its original form, indicating a reversible transition. Equally important, we show that the force-induced conformation exposes hidden epitopes previously buried in the Tfp fiber. We postulate that this transition provides a means for N. gonorrhoeae to maintain attachment to its host while withstanding intermittent forces encountered in the environment. Our findings demonstrate the need to reassess our understanding of Tfp dynamics and functions. They could also explain the structural diversity of other helical polymers while presenting a unique mechanism for polymer elongation and exemplifying the extreme structural plasticity of biological polymers.

Hummer and Szabo-like Potential of Mean Force Estimator for Bidirectional Nonequilibrium Pulling Experiments/Simulations

Paolo Nicolini, Piero Procacci and Riccardo Chelli

In the framework of single-molecule pulling experiments, the system is typically driven out of equilibrium by a time-dependent external potential V(t) acting on a collective coordinate such that the total Hamiltonian is the sum of V(t) and the Hamiltonian in the absence of external perturbation. Nonequilibrium work theorems such as Jarzynski equality and Crooks fluctuation theorem have been devised to recover free energy differences between states of this extended system. However, one is often more interested in the potential of mean force of the unperturbed Hamiltonian, i.e., in the effective potential dictating the equilibrium distribution of the collective coordinate in the absence of the external potential. In this respect, Hummer and Szabo proposed an algorithm to estimate the desired free energy differences when pulling experiments are performed in only one direction of the process (Proc. Natl. Acad. Sci. USA 2001, 98, 3658). In this paper, we present a potential of mean force estimator of the unperturbed system that exploits the work measured in both forward and backward directions of the process. The method is based on the reweighting technique of Hummer and Szabo and on the Bennett acceptance ratio. Using Brownian-dynamics simulations on a double-well free energy surface, we show that the estimator works satisfactorily in any pulling situation, from nearly equilibrium to strongly dissipative regimes. The method is also applied to the unfolding/refolding process of decaalanine, a system vastly used to illustrate and to test nonequilibrium methodologies. A thorough comparative analysis with another bidirectional potential of mean force estimator (Minh, D. D. L.; Adib, A. B. Phys. Rev. Lett. 2008, 100, 180602) is also presented. The proposed approach is well-suited to recover free energy profiles from single-molecule bidirectional-pulling experiments such as those performed by optical tweezers or atomic force microscopes.


Single-step replication of a highly integrated PDMS optofluidic analysis system

Martin Amberg, Sebastian Stoebenau, and Stefan Sinzinger

Micromilling is a promising technology for the fabrication of surface profiles with optical quality. We present a highly integrated optofluidic system made of polydimethylsiloxane (PDMS). The system is replicated in a single-step process from a micromilled polymethyl methacrylate master mold. It already includes the reservoirs, the channel system, as well as the optical interconnect surfaces for high numerical aperture objectives. We demonstrate the potential of this approach by laser-based three-dimensional optical manipulation within the replicated system. To our knowledge, this is the first time that a PDMS membrane is used as a well-defined channel wall for an optical trapping setup.


Tuesday, August 10, 2010

Dynamic axial stabilization of counter-propagating beam-traps with feedback control

Sandeep Tauro, Andrew Bañas, Darwin Palima, and Jesper Glückstad

Optical trapping in a counter-propagating (CP) beam-geometry provides unique advantages in terms of working distance, aberration requirements and intensity hotspots. However, its axial performance is governed by the wave propagation of the opposing beams, which can limit the practical geometries. Here we propose a dynamic method for controlling axial forces to overcome this constraint. The technique uses computer-vision object tracking of the axial position, in conjunction with software-based feedback, for dynamically stabilizing the axial forces. We present proof-of-concept experiments showing real-time rapid repositioning coupled with a strongly enhanced axial trapping for a plurality of particles of varying sizes. We also demonstrate the technique’s adaptability for real-time reconfigurable feedback-trapping of a dynamically growing structure that mimics a continuously dividing cell colony. Advanced implementation of this feedback-driven approach can help make CP-trapping resistant to a host of perturbations such as laser fluctuations, mechanical vibrations and other distortions emphasizing its experimental versatility.


Optical Manipulation with Planar Silicon Microring Resonators

Shiyun Lin, Ethan Schonbrun and Kenneth Crozier

We demonstrate optically trapping of microparticles on silicon microring resonators. Once trapped on a microring, a particle can be confined in an optical potential with a depth of 25kBT over the entire microring’s circumference. The particles are propelled around the microring at hundreds of micrometers per second, producing periodic revolutions at a few hertz. We anticipate that the increased force and highly accurate positioning obtainable with this system will lead to various nanomanipulation applications.


Monday, August 9, 2010

Simulation of single-molecule trapping in a nanochannel

William Neil Robinson and Lloyd M. Davis

The detection and trapping of single fluorescent molecules in solution within a nanochannel is studied using numerical simulations. As optical forces are insufficient for trapping molecules much smaller than the optical wavelength, a means for sensing a molecule's position along the nanochannel and adjusting electrokinetic motion to compensate diffusion is assessed. Fluorescence excitation is provided by two adjacently focused laser beams containing temporally interleaved laser pulses. Photon detection is time-gated, and the displacement of the molecule from the middle of the two foci alters the count rates collected in the two detection channels. An algorithm for feedback control of the electrokinetic motion in response to the timing of photons, to reposition the molecule back toward the middle for trapping and to rapidly reload the trap after a molecule photobleaches or escapes, is evaluated. While accommodating the limited electrokinetic speed and the finite latency of feedback imposed by experimental hardware, the algorithmis shown to be effective for trapping fast-diffusing single-chromophore molecules within a micron-sized confocal region. Studies show that there is an optimum laser power for which loss of molecules from the trap due to either photobleaching or shot-noise fluctuations isminimized.


Phase contrast optical tweezers

Ali Mahmoudi and S. Nader S. Reihani

In this paper, for the first time, we report on systematic theoretical and experimental investigation of Phase Contrast Optical Tweezers (PCOT) which could be an indispensable tool for micromanipulation of the transparent micro and nano objects such as biological tissues and vesicles. The quadrant photodiode detection scheme and the power-spectrum calibration method is shown to be valid for this case. We have shown that the phase objective with new designed phase plates can provide nearly aberration-free condition at a desired depth. This could be a valuable advantage for simultaneous in-depth micro-manipulations and visualization of the sample.


Electrochemical Characterization of a Single Electricity-Producing Bacterial Cell of Shewanella by Using Optical Tweezers

Huan Liu Dr., Greg J. Newton, Ryuhei Nakamura Dr., Kazuhito Hashimoto Prof. Dr., Shuji Nakanishi Dr.

Members of genus Shewanella are Gram-negative bacteria that can utilize solid-state metal oxide as a terminal electron acceptor for respiration. The direct electrical connection between a single cell and a microelectrode is characterized by an optical tweezers technique.


A clustered speckle approach to optical trapping

J.P. Staforelli, J.M. Brito, E. Vera, P. Solano and A. Lencina

An in situ study of the clustered speckle 3D structure using an optical tweezer setup is presented. Clustered speckles appear when a coherently illuminated diffuser is imaged through a pupil mask with several apertures, properly distributed over a closed path, which is placed before the objective lens of a standard optical trapping system. Thus, light volumes are reduced several times when compared with standard speckles, being even smaller than the focus volume of a Gaussian beam commonly used to trap. Moreover, clustered speckles have odd statistical properties which differentiated it from standard speckles. Then, geometrically ordered multiple trapping arrays with statistical random distribution of intensities can be created with this technique. This fact could enable different studies concerning optical binding or new developments in coherent matter wave transport where Optical Trapping has been proven with standard speckles. In this work, a qualitative analysis of clustered speckles in an optical tweezer setup relative to the number of apertures in the mask and their size is carried on. Besides, in the Rayleigh regime, a general quantitative method to characterize the trapping capability of an optical field is proposed. Then, it is applied to clustered speckles. As a result, a relation between aperture size and the maximum size of the particles that could be trapped is found. This fact opens the possibility of engineering the statistic of the trapped particles by properly selecting the pupil mask.


Friday, August 6, 2010

Linear diode laser bar optical stretchers for cell deformation

Ihab Sraj, David W. M. Marr, and Charles D. Eggleton

To investigate the use of linear diode laser bars to optically stretch cells and measure their mechanical properties, we present numerical simulations using the immersed boundary method (IBM) coupled with classic ray optics. Cells are considered as three-dimensional (3D) spherical elastic capsules immersed in a fluid subjected to both optical and hydrodynamic forces in a periodic domain. We simulate cell deformation induced by both single and dual diode laser bar configurations and show that a single diode laser bar induces significant stretching but also induces cell translation of speed < 10 µm/sec for applied 6.6 mW/µm power in unconfined systems. The dual diode laser bar configuration, however, can be used to both stretch and optically trap cells at a fixed position. The net cell deformation was found to be a function of the total laser power and not the power distribution between single or dual diode laser bar configurations.


Compact interferometric optical tweezer for patterned trapping and manipulation of polystyrene spheres and SWCNTs

Ranjeet Kumar; Chandra Shakher; Dalip Singh Mehta

We demonstrate a simple and compact optical interferometric unit combined with a conventional optical tweezer system for simultaneous multiple trapping and micromanipulation of mono-dispersed polystyrene spheres and aggregation of small-floating clusters of single-walled carbon nanotubes (SWCNTs). The interferometric unit was made compact by means of coating a thin layer of aluminum oxide on one side of the cubic beam splitter (CBS) which works as a static reference mirror and an adjustable mirror was mounted on a XYZ translational stage facing the other adjacent side of the CBS. Thus, the developed interferometric unit is quite analogous to a Michelson interferometer but is compact. Sinusoidal interference fringes with variable carrier frequency and orientations were generated. The interference fringes were then used for multiple trapping of polystyrene spheres along bright fringes resulting in pattern formation and also the aggregation of tiny floating clusters of SWCNTs. The proposed system is compact and easy to align because of its common-path geometry.


Thursday, August 5, 2010

Biophotonic techniques for the study of malaria-infected red blood cells

Jakob M. A. Mauritz, Alessandro Esposito, Teresa Tiffert, Jeremy N. Skepper, Alice Warley, Young-Zoon Yoon, Pietro Cicuta, Virgilio L. Lew, Jochen R. Guck and Clemens F. Kaminski

Investigation of the homeostasis of red blood cells upon infection by Plasmodium falciparum poses complex experimental challenges. Changes in red cell shape, volume, protein, and ion balance are difficult to quantify. In this article, we review a wide range of optical techniques for quantitative measurements of critical homeostatic parameters in malaria-infected red blood cells. Fluorescence lifetime imaging and tomographic phase microscopy, quantitative deconvolution microscopy, and X-ray microanalysis, are used to measure haemoglobin concentration, cell volume, and ion contents. Atomic force microscopy is briefly reviewed in the context of these optical methodologies. We also describe how optical tweezers and optical stretchers can be usefully applied to empower basic malaria research to yield diagnostic information on cell compliance changes upon malaria infection. The combined application of these techniques sheds new light on the detailed mechanisms of malaria infection providing potential for new diagnostic or therapeutic approaches.


Probing oxidative stress in single erythrocytes with Raman Tweezers

E. Zachariah, A. Bankapur, C. Santhosh, M. Valiathan and D. Mathur

Raman Tweezers have been successfully applied to characterize chemically-induced oxidative stress on optically-trapped live, single erythrocytes. There is significant enhancement in Raman peak intensities corresponding to S=S and C–S stretching modes that are induced by oxidative stress. This is consistent with the formation of mixed disulphides between protein SH groups and low-molecular-mass thiols such as glutathione during oxidative damage to cells. Enhancement in glutathione level as a protective response against oxidative stress has been observed. Principal component analysis of the data yields good discrimination between spectra of normal and stress-induced red blood cells.

Tunable optical gradient trap by radial varying polarization Bessel-Gauss beam

Gao, X.-M., Hu, S., Li, J.-S., Ding, Z.-H., Guo, H.-M., Zhuang, S.-L.

Optical tweezers play an important role in many domains, especially in life science. And optical gradient force is necessary for constructing optical tweezers. In this paper, the optical gradient force in the focal region of radial varying polarization Bessel- Gauss beam is investigated numerically by means of vector diffraction theory. Results show that the beam parameter and vary rate parameter that indicates the change speed of polarization rotation angle affect the optical gradient force pattern very considerably, and some novel force distributions may come into being, such as multiple force minimums, force ring, and force crust. Therefore, the focusing of radial varying polarization Bessel-Gauss beam can be used to construct optical traps.


Membrane nanotubes drawn by optical tweezers transmit electrical signals between mammalian cells over long distances

Pedro Pascoal, Davor Kosanic, Marinela Gjoni and Horst Vogel

Biological cells continuously change shape allowing essential functions such as cell motility, vesicle-mediated release/uptake of soluble and membrane components or nanotube-mediated cell–cell communications. Here we use single cell micromanipulation to induce functional changes of cell shape for nanobiotechnological applications. Optical tweezers are focused on the plasma membrane of living cells to pull membrane nanotubes of 200 nanometre diameters and 100 micrometre lengths. Upon switching off the laser tweezer membrane nanotubes relax back to the cell surface. Single-exponential relaxation times deliver local mechanical properties of cells' plasma membrane. Nanotubes pulled beyond 100 micrometre tear off and form micrometre-sized vesicles carrying functional membrane receptors and cytoplasmic signaling components. Membrane nanotubes from one cell can be contacted to adjacent cells forming via connexins intercellular electrical connections within seconds in all directions. Our method opens broad applications for multiplexing single-cell analytics to submicrometer/subfemtoliter ranges and for creating artificial intercellular signaling networks, both not attainable by current methodologies.


TweezPal – Optical tweezers analysis and calibration software

Natan Osterman

Optical tweezers, a powerful tool for optical trapping, micromanipulation and force transduction, have in recent years become a standard technique commonly used in many research laboratories and university courses. Knowledge about the optical force acting on a trapped object can be gained only after a calibration procedure which has to be performed (by an expert) for each type of trapped objects. In this paper we present TweezPal, a user-friendly, standalone Windows software tool for optical tweezers analysis and calibration. Using TweezPal, the procedure can be performed in a matter of minutes even by non-expert users. The calibration is based on the Brownian motion of a particle trapped in a stationary optical trap, which is being monitored using video or photodiode detection. The particle trajectory is imported into the software which instantly calculates position histogram, trapping potential, stiffness and anisotropy.


Monitoring of laser micromanipulated optically trapped cells by digital holographic microscopy

Björn Kemper, Patrik Langehanenberg, Alexander Höink, Gert von Bally, Falk Wottowah, Stefan Schinkinger, Jochen Guck, Josef Käs, Ilona Bredebusch, Jürgen Schnekenburger, Karin Schütze

For a precise manipulation of particles and cells with laser light as well as for the understanding and the control of the underlying processes it is important to visualize and quantify the response of the specimens. Thus, we investigated if digital holographic microscopy (DHM) can be used in combination with microfluidics to observe optically trapped living cells in a minimally invasive fashion during laser micromanipulation. The obtained results demonstrate that DHM multi-focus phase contrast provides label-free quantitative monitoring of optical manipulation with a temporal resolution of a few milliseconds.


Insights into the mechanisms of myosin and kinesin molecular motors from the single-molecule unbinding force measurements

Sergey V. Mikhailenko, Yusuke Oguchi and Shin'ichi Ishiwata

In cells, ATP (adenosine triphosphate)-driven motor proteins, both cytoskeletal and nucleic acid-based, operate on their corresponding ‘tracks’, that is, actin, microtubules or nucleic acids, by converting the chemical energy of ATP hydrolysis into mechanical work. During each mechanochemical cycle, a motor proceeds via several nucleotide states, characterized by different affinities for the ‘track’ filament and different nucleotide (ATP or ADP) binding kinetics, which is crucial for a motor to efficiently perform its cellular functions. The measurements of the rupture force between the motor and the track by applying external loads to the individual motor–substrate bonds in various nucleotide states have proved to be an important tool to obtain valuable insights into the mechanism of the motors' performance. We review the application of this technique to various linear molecular motors, both processive and non-processive, giving special attention to the importance of the experimental geometry.

Two-dimensional dielectrophoretic particle trapping in a hybrid crystal/PDMS-system

Michael Esseling, Frank Holtmann, Mike Woerdemann, and Cornelia Denz

Dielectrophoretic forces originating from highly modulated electric fields can be used to trap particles on surfaces. An all-optical way to induce such fields is the use of a photorefractive material, where the fields that modulate the refractive index are present at the surface. We present a method for two-dimensional particle alignment on an optically structured photorefractive lithium niobate crystal. The structuring is done using an amplitude-modulating spatial light modulator and laser illumination. We demonstrate trapping of uncharged graphite particles in periodic and arbitrary patterns and provide a discussion of the limitations and the necessary boundary conditions for maximum trapping efficiency. The photorefractive crystal is utilized as bottom part of a PDMS channel in order to demonstrate two-dimensional dielectrophoretic trapping in a microfluidic system.


Single cell optical transfection

David J. Stevenson, Frank J. Gunn-Moore, Paul Campbell and Kishan Dholakia

The plasma membrane of a eukaryotic cell is impermeable to most hydrophilic substances, yet the insertion of these materials into cells is an extremely important and universal requirement for the cell biologist. To address this need, many transfection techniques have been developed including viral, lipoplex, polyplex, capillary microinjection, gene gun and electroporation. The current discussion explores a procedure called optical injection, where a laser field transiently increases the membrane permeability to allow species to be internalized. If the internalized substance is a nucleic acid, such as DNA, RNA or small interfering RNA (siRNA), then the process is called optical transfection. This contactless, aseptic, single cell transfection method provides a key nanosurgical tool to the microscopist—the intracellular delivery of reagents and single nanoscopic objects. The experimental possibilities enabled by this technology are only beginning to be realized. A review of optical transfection is presented, along with a forecast of future applications of this rapidly developing and exciting technology.