Thursday, February 28, 2013

Effects of Plasma Membrane Cholesterol Level and Cytoskeleton F-Actin on Cell Protrusion Mechanics

Nima Khatibzadeh, Alexander A. Spector, William E. Brownell, Bahman Anvari

Protrusions are deformations that form at the surface of living cells during biological activities such as cell migration. Using combined optical tweezers and fluorescent microscopy, we quantified the mechanical properties of protrusions in adherent human embryonic kidney cells in response to application of an external force at the cell surface. The mechanical properties of protrusions were analyzed by obtaining the associated force-length plots during protrusion formation, and force relaxation at constant length. Protrusion mechanics were interpretable by a standard linear solid (Kelvin) model, consisting of two stiffness parameters, k0 and k1 (with k0>k1), and a viscous coefficient. While both stiffness parameters contribute to the time-dependant mechanical behavior of the protrusions, k0 and k1 in particular dominated the early and late stages of the protrusion formation and elongation process, respectively. Lowering the membrane cholesterol content by 25% increased the k0 stiffness by 74%, and shortened the protrusion length by almost half. Enhancement of membrane cholesterol content by nearly two-fold increased the protrusion length by 30%, and decreased the k0 stiffness by nearly two-and-half-fold as compared with control cells. Cytoskeleton integrity was found to make a major contribution to protrusion mechanics as evidenced by the effects of F-actin disruption on the resulting mechanical parameters. Viscoelastic behavior of protrusions was further characterized by hysteresis and force relaxation after formation. The results of this study elucidate the coordination of plasma membrane composition and cytoskeleton during protrusion formation.


[Conference] Optical Trapping and Optical Micromanipulation X

Dear Blog members,

Abstracts for OTOM X, the Optical Trapping & Optical Micromanipulation Conference can still be submitted, through Monday, March 11: http://tinyurl.com/cef4vgv

Axial potential mapping of optical tweezers for biopolymer stretching: the bead size matters

Arash Ahmadi and S. Nader S. Reihani
Optical tweezers (OT) are widely used for pico (and femto)-Newton range force measurements. The appropriate choice of the bead size is not well understood for biopolymer stretching applications of OT. We have shown, both by theory and experiment, that wrong choice of the bead size could cause errors as large as 295% in the measured force. We provide a simple map for correct choice of the bead size and the direction of pulling for such applications. There is a good agreement between our theoretical and experimental results.


Subwavelength optical trapping with a fiber-based surface plasmonic lens

Yuxiang Liu, Felix Stief, and Miao Yu

We demonstrate three-dimensional (3D) optical trapping of subwavelength polystyrene beads and bacteria with a surface plasmonic lens fabricated on the endface of an optical fiber. To the best of our knowledge, this is the first demonstration of 3D trapping of subwavelength particles with single fiber optical tweezers. The optical power for achieving a stable 3D trap is smaller compared with conventional optical tweezers, indicating a stronger trap. Compared with surface plasmon tweezers, the trap enabled by our fiber tweezers is located ≈6 wavelengths away from the fiber endface, reducing thermal effects due to the metal absorption and preventing physical contact with the trapped objects.

HoloHands: games console interface for controlling holographic optical manipulation

C McDonald, M McPherson, C McDougall and D McGloin
The increasing number of applications for holographic manipulation techniques has sparked the development of more accessible control interfaces. Here, we describe a holographic optical tweezers experiment which is controlled by gestures that are detected by a Microsoft Kinect. We demonstrate that this technique can be used to calibrate the tweezers using the Stokes drag method and compare this to automated calibrations. We also show that multiple particle manipulation can be handled. This is a promising new line of research for gesture-based control which could find applications in a wide variety of experimental situations.


Optically Driven Gear-Based Mechanical Microtransducer for a Lab-on-a-Chip

Lee, Yi-Hsiung; Liu, Yi-Jui; Tzou, Ching-Fu; Bouriau, Michel; Baldeck, Patrice L.; Lin, Chih-Lang
In this study, we propose an optically driven gear-based microtransducer composed of a mechanical arm, a sphere, and two gears, which are fabricated by two-photon polymerization technique. Optical tweezers is employed to grasp the sphere as a force exerted point to manipulate the mechanical arm. The two gears with different diameters to form a required gear ratio are meshed for the tranducing demonstration. Thanks to the classic lever function, the mechanical arm can multiply the optical tweezers force as the request of tranducing performance. The experimental result indicates that the transducer enables precise applied forces and the directions of the microtransducer by optical tweezers at micron scale. The ratio of rotational angles has good agreement with the gear ratio as the classic gear function. Such gear-based optically driven mechanical transducer provides a possibility for driving micron-sized complex mechanisms, which is expected to perform as a mechanical operator in micro-channels for the applications of the “Lab-on-a-chip.“

Mechanical characteristics of human red blood cell membrane change due to C60 nanoparticle infiltration

Xiaoyue Zhang , Yong Zhang , Yue Zheng and Biao Wang

The mechanical characteristics of human red blood cell (RBC) membrane change due to C60 nanoparticle (NP) infiltration have been investigated in the present work. Using experimental approaches, including optical tweezer (OT) stretching and atomic force microscopy (AFM) indentation, we found that RBCs in the presence of C60 NPs are softer than normal RBCs. The strain–stress relations of both normal and C60 infiltrated RBC membranes are extracted from the data of AFM indentation, from which we proved that C60 NP infiltration can affect the mechanical properties of RBC membrane and tend to weaken the tensile resistance of lipids bilayers. In order to explain this experimental phenomenon, a mechanical model has been developed. Based on this model, the strain–stress relations of both normal and C60 infiltrated lipid bilayers are calculated with consideration of intermolecular interactions. The theoretical results are in great agreement with the experimental results. The influence of C60 NP concentration on the mechanical properties of RBC membrane is successfully predicted. Higher concentrations of C60 NPs in the lipid bilayers will lead to increased damage to the cell membrane, implying that the dosage of C60 NPs should be controlled in medical applications.

Tuesday, February 26, 2013

New Technique for Single-Beam Gradient-Force Laser Trapping in Air

Masaki Michihata, Tada-aki Yoshikane, Terutake Hayashi and Yasuhiro Takaya

Laser trapping is becoming an important technique for microsystem technologies. To apply it to industrial uses, it should be developed to function in air. However, there is not much research about laser trapping in air. One of the reasons is the difficulty of trapping micro-objects. Therefore, we have proposed a new technique to trap micro-objects in air. In particular, we focused our attention on the substrate where the micro-object is set. By applying a textured surface and employing tungsten carbide as the substrate material, the trapping probability was improved by a remarkable amount. Although a certain degree of improvement was attained, the trapping was still not perfect. To ascertain the forces working on the micro-object, an analytical calculation of the adhesion forces and an electromagnetic simulation of the optical forces were implemented. Based on these calculations, we discuss what the most important factor is for successful laser trapping in air.

Design and Two-Photon Polymerization of Complex Functional Micro-Objects for Lab-on-a-Chip: Rotating Micro-Valves

Chung, Tien-Tung; Tseng, Chang-Li; Hung, Chia-Ping; Lin, Chih-Lang; Baldeck, Patrice L.
Two-Photon Polymerization is a powerful technology that can be used to fabricate complex functional micro-objects in lab-on-a-chip platforms. It is a laser-based prototyping technique with full tri-dimensional capability and sub-micron resolution. We report on the development of a computer-assisted design and fabrication process based on Q-switched Nd:YAG microlasers and AutoCAD environment. Microlasers with sub-nanosecond pulses of visible light are efficient and low cost lasers for two-photon microfabrication. Polymerization is easily obtained with sub-milliwatt average powers (12 kHz repetition rate). The microfabrication software has been developed to design the micro-object models and to calculate their laser trajectories using AutoCAD. It is open platform for the design of 3D solids with full access to entities information, and an efficient slicing command that can cut solids from any orientation plane. As a typical example of complex functional micro-object, we report on the fabrication of laser-driven rotating micro-valves. The design parameters have been optimized to allow a free movement of the valve around its axis using the optical force of a laser tweezers.

Monday, February 25, 2013

Integrated microfluidic device for single-cell trapping and spectroscopy

C. Liberale, G. Cojoc, F. Bragheri, P. Minzioni, G. Perozziello, R. La Rocca, L. Ferrara, V. Rajamanickam, E. Di Fabrizio, and I. Cristiani
Optofluidic microsystems are key components towards lab-on-a-chip devices for manipulation and analysis of biological specimens. In particular, the integration of optical tweezers (OT) in these devices allows stable sample trapping, while making available mechanical, chemical and spectroscopic analyses.

Transition of a particle between adjacent optical traps: A study using catastrophe theory

Deepak Kumar, Shankar Ghosh, and S. BhattacharyaIn spite of the widespread use of optical tweezers as a quantitative tool to measure small forces, there exists no unambiguous and simple experimental method for either validating its theoretically predicted form or empirically parameterizing it over the entire range. This problem is addressed by studying the transition of a colloidal particle between two spatially separated optical traps. The transition as a function of the relative intensity of the traps and the separation between them reveals a formal resemblance to the “butterfly catastrophe” which also maps onto to phase transitions observed, for example, in ferroelectrics on a phenomenological level. The method has been used to experimentally determine the force-displacement curve for an optical trap over its entire range.

Measuring red blood cell aggregation forces using double optical tweezers

Heloise P. Fernandes, Adriana Fontes, André Thomaz, Vagner Castro, Carlos L. Cesar and Maria L. Barjas-Castro
Classic immunohematology approaches, based on agglutination techniques, have been used in manual and automated immunohematology laboratory routines. Red blood cell (RBC) agglutination depends on intermolecular attractive forces (hydrophobic bonds, Van der Walls, electrostatic forces and hydrogen bonds) and repulsive interactions (zeta potential). The aim of this study was to measure the force involved in RBC aggregation using double optical tweezers, in normal serum, in the presence of erythrocyte antibodies and associated to agglutination potentiator solutions (Dextran, low ionic strength solution [LISS] and enzymes). The optical tweezers consisted of a neodymium:yattrium aluminium garnet (Nd:YAG) laser beam focused through a microscope equipped with a minicam, which registered the trapped cell image in a computer where they could be analyzed using a software. For measuring RBC aggregation, a silica bead attached to RBCs was trapped and the force needed to slide one RBC over the other, as a function of the velocities, was determined. The median of the RBC aggregation force measured in normal serum (control) was 1 × 10−3 (0.1–2.5) poise.cm. The samples analyzed with anti-D showed 2 × 10−3 (1.0–4.0) poise.cm (p < 0.001). RBC diluted in potentiator solutions (Dextran 0.15%, Bromelain and LISS) in the absence of erythrocyte antibodies, did not present agglutination. High adherence was observed when RBCs were treated with papain. Results are in agreement with the imunohematological routine, in which non-specific results are not observed when using LISS, Dextran and Bromelain. Nevertheless, false positive results are frequently observed in manual and automated microplate analyzer using papain enzyme. The methodology proposed is simple and could provide specific information with the possibility of meansuration regarding RBC interaction.


Thursday, February 21, 2013

Simulation of a Brownian particle in an optical trap

Giorgio Volpe and Giovanni Volpe
An optically trapped Brownian particle is a sensitive probe of molecular and nanoscopic forces. An understanding of its motion, which is caused by the interplay of random and deterministic contributions, can lead to greater physical insight into the behavior of stochastic phenomena. The modeling of realistic stochastic processes typically requires advanced mathematical tools. We discuss a finite difference algorithm to compute the motion of an optically trapped particle and the numerical treatment of the white noise term. We then treat the transition from the ballistic to the diffusive regime due to the presence of inertial effects on short time scales and examine the effect of an optical trap on the motion of the particle. We also outline how to use simulations of optically trapped Brownian particles to gain understanding of nanoscale force and torque measurements, and of more complex phenomena, such as Kramers transitions, stochastic resonant damping, and stochastic resonance.

Double nanohole optical trapping: Dynamics and protein-antibody co-trapping

Ana Zehtabi-Oskuie , Hao Jiang , Bryce Cyr , Douglas Rennehan, Ahmed Al-Balushi and Reuven GordonA double nanohole in a metal film can optically trap nanoparticles such as polystyrene/silica spheres, encapsulated quantum dots and up-converting nanoparticles. Here we study the dynamics of trapped particles, showing a skewed distribution and low roll-off frequency that are indicative of Kramers-hopping at the nanoscale. Numerical simulations of trapped particles show a double-well potential normally found in Kramers-hopping systems, as well as providing quantitative agreement with the overall trapping potential. In addition, we demonstrate co-trapping of bovine serum albumin (BSA) with anti-BSA by sequential delivery in a microfluidic channel. This co-trapping opens up exciting possibilities for the study of protein interactions at the single particle level.

Optical trapping Rayleigh dielectric spheres with focused anomalous hollow beams

Zhirong Liu and Daomu ZhaoFocusing properties of anomalous hollow beams (AHBs) are theoretically and numerically investigated. The radiation forces acting upon a Rayleigh dielectric sphere produced by focused AHBs are also studied. Results show that focused AHBs can be used to trap and manipulate microsized dielectric spheres with high-refractive index at the focal point. Finally, the stability conditions for effective trapping particles are analyzed in detail. The results presented here are of interest in some possible applications by making use of AHBs.

Automated Cell Transport in Optical Tweezers-Assisted Microfluidic Chambers

Svec, P. ; Wang, C. ; Seale, K. T. ; Wikswo, J. P. ; Losert, W.; Gupta, S. K.

In this paper, we present a physics-aware, planning approach for automated transport of cells in an optical tweezers-assisted microfluidic chamber. The approach can be used for making a uniform distribution of cells inside the chamber to allow the study of a variety of biological processes, including cell signaling. Fluid forces inside the chamber, modeled using computational fluid dynamics, are incorporated into the widely used Langevin equation to simulate the motion of cells. The developed simulator was used for building a map that contains probabilities of a cell successfully reaching one of the outlets of the chamber from different locations under the influence of the fluid flow. The developed planner not only generates collision-free paths that exploit the fluid flow inside the chamber but also utilizes the offline generated simulation data to decide suitable locations for releasing the cells. This ensures fast and robust cell transport, while minimizing the required laser power and operational time. The planner is based on the heuristic D* Lite algorithm that employs a specific cost function for searching over a novel state-action space representation. The effectiveness of the planning algorithm is demonstrated using both simulation and physical experiments in a microfluidics-optical tweezers hybrid manipulation setup.


Simultaneous trapping of low- and high-index microparticles by using highly focused elegant Hermite-cosh-Gaussian beams

Zhirong Liu, Kaikai Huang, Daomu Zhao

The analytical expression for the propagation of elegant Hermite-cosh-Gaussian (EHCHG) beams through a paraxial ABCD optical system is derived and used to study the radiation forces produced by highly focused EHCHG beams acting on a Rayleigh dielectric particle. Owing to the characteristics of this kind of beams our analysis shows that it can be expected to simultaneously trap and manipulate dielectric spheres with low-refractive index at the focal point, and particles with high-refractive index nearby the focal point. Finally, the stability conditions for effective trapping and manipulating particles by this kind of focused beams are discussed.

Wednesday, February 20, 2013

A focal spot with variable intensity distribution for optical tweezers

X Hao, C F Kuang, Y H Li and X Liu
An optical setup that can generate a focused light spot, with its shape tunable from a very sharp peak-centered beam to a donut-shaped dark-centered beam, is always desired in optical tweezing. This setup is now realized by incorporating an image inverting interferometer (III) and a helical phase element into a radially polarized beam generator, which enables the trapping and control of a large variety of particles with a single optical tweezing system. The performance of this system is investigated theoretically.

Exploring protein-DNA interactions in 3D using in situ construction, manipulation and visualization of individual DNA dumbbells with optical traps, microfluidics and fluorescence microscopy

Anthony L Forget, Christopher C Dombrowski, Ichiro Amitani & Stephen C Kowalczykowski

In this protocol, we describe a procedure to generate 'DNA dumbbells'—single molecules of DNA with a microscopic bead attached at each end—and techniques for manipulating individual DNA dumbbells. We also detail the design and fabrication of a microfluidic device (flow cell) used in conjunction with dual optical trapping to manipulate DNA dumbbells and to visualize individual protein-DNA complexes by single-molecule epifluorescence microscopy. Our design of the flow cell enables the rapid movement of trapped molecules between laminar flow channels and a flow-free reservoir. The reservoir provides the means to examine the formation of protein-DNA complexes in solution in the absence of external flow forces while maintaining a predetermined end-to-end extension of the DNA. These features facilitate the examination of the role of 3D DNA conformation and dynamics in protein-DNA interactions. Preparation of flow cells and reagents requires 2 days each; in situ DNA dumbbell assembly and imaging of single protein-DNA complexes require another day.


Orthogonal optical force separation simulation of particle and molecular species mixtures under direct current electroosmotic driven flow for applications in biological sample preparation

Sarah J. R. Staton, Alex Terray, Greg E. Collins, Sean J. Hart

Presented here are the results from numerical simulations applying optical forces orthogonally to electroosmotically induced flow containing both molecular species and particles. Simulations were conducted using COMSOL v4.2a Multiphysics® software including the particle tracking module. The study addresses the application of optical forces to selectively remove particulates from a mixed sample stream that also includes molecular species in a pinched flow microfluidic device. This study explores the optimization of microfluidic cell geometry, magnitude of the applied direct current electric field, electroosmotic flow rate, diffusion, and magnitude of the applied optical forces. The optimized equilibrium of these various contributing factors aids in the development of experimental conditions and geometry for future experimentation as well as directing experimental expectations, such as diffusional losses, separation resolution, and percent yield. The result of this work generated an optimized geometry with flow conditions leading to negligible diffusional losses of the molecular species while also being able to produce particle removal at near 100% levels. An analytical device, such as the one described herein with the capability to separate particulate and molecular species in a continuous, high-throughput fashion would be valuable by minimizing sample preparation and integrating gross sample collection seamlessly into traditional analytical detection methods.


Quantum Dot-Based Thermal Spectroscopy and Imaging of Optically Trapped Microspheres and Single Cells

Patricia Haro-González, William T. Ramsay, Laura Martinez Maestro, Blanca del Rosal, Karla Santacruz-Gomez, Maria del Carmen Iglesias-de la Cruz, Francisco Sanz-Rodríguez, Jing Yuang Choo, Paloma Rodriguez Sevilla, Marco Bettinelli, Debaditya Choudhury, Ajoy K. Kar, José García Solé, Daniel Jaque, Lynn Paterson

Laser-induced thermal effects in optically trapped microspheres and single cells are investigated by quantum dot luminescence thermometry. Thermal spectroscopy has revealed a non-localized temperature distribution around the trap that extends over tens of micrometers, in agreement with previous theoretical models besides identifying water absorption as the most important heating source. The experimental results of thermal loading at a variety of wavelengths reveal that an optimum trapping wavelength exists for biological applications close to 820 nm. This is corroborated by a simultaneous analysis of the spectral dependence of cellular heating and damage in human lymphocytes during optical trapping. This quantum dot luminescence thermometry demonstrates that optical trapping with 820 nm laser radiation produces minimum intracellular heating, well below the cytotoxic level (43 °C), thus, avoiding cell damage.

Monday, February 18, 2013

Fundamental constraints on particle tracking with optical tweezers

Michael A Taylor, Joachim Knittel and Warwick P Bowen
A general quantum limit to the sensitivity of particle position measurements is derived following the simple principle of the Heisenberg microscope. The value of this limit is calculated for particles in the Rayleigh and Mie scattering regimes, and with parameters which are relevant to optical tweezers experiments. The minimum power required to observe the zero-point motion of a levitating bead is also calculated, with the optimal particle diameter always smaller than the wavelength. We show that recent optical tweezers experiments are within two orders of magnitude of quantum limited sensitivity, suggesting that quantum optical resources may soon play an important role in high sensitivity tracking applications.

Assembly and control of 3D nematic dipolar colloidal crystals

A. Nych, U. Ognysta, M. Škarabot, M. Ravnik, S. Žumer & I. Muševič

Topology has long been considered as an abstract mathematical discipline with little connection to material science. Here we demonstrate that control over spatial and temporal positioning of topological defects allows for the design and assembly of three-dimensional nematic colloidal crystals, giving some unexpected material properties, such as giant electrostriction and collective electro-rotation. Using laser tweezers, we have assembled three-dimensional colloidal crystals made up of 4 μm microspheres in a bulk nematic liquid crystal, implementing a step-by-step protocol, dictated by the orientation of point defects. The three-dimensional colloidal crystals have tetragonal symmetry with antiparallel topological dipoles and exhibit giant electrostriction, shrinking by 25–30% at 0.37 V μm−1. An external electric field induces a reversible and controllable electro-rotation of the crystal as a whole, with the angle of rotation being ~30° at 0.14 V μm−1, when using liquid crystal with negative dielectric anisotropy. This demonstrates a new class of electrically highly responsive soft materials.

Resolving Stable Axial Trapping Points of Nanowires in an Optical Tweezers Using Photoluminescence Mapping

Fan Wang, Wen Jun Toe, Woei Ming Lee, David McGloin, Qiang Gao, Hark Hoe Tan, Chennupati Jagadish, and Peter J. Reece
Axially resolved microphotoluminescence mapping of semiconductor nanowires held in an optical tweezers reveals important new experimental information regarding equilibrium trapping points and trapping stability of high aspect ratio nanostructures. In this study, holographic optical tweezers are used to scan trapped InP nanowires along the beam direction with respect to a fixed excitation source and the luminescent properties are recorded. It is observed that nanowires with lengths on the range of 3–15 μm are stably trapped near the tip of the wire with the long segment positioned below the focus in an inverted trapping configuration. Through the use of trap multiplexing we investigate the possibility of improving the axial stability of the trapped nanowires. Our results have important implication for applications of optically assisted nanowire assembly and optical tweezers based scanning probes microscopy.

Structural and mechanical properties of individual human telomeric G-quadruplexes in molecularly crowded solutions

Soma Dhakal, Yunxi Cui, Deepak Koirala, Chiran Ghimire, Saurabh Kushwaha, Zhongbo Yu, Philip M. Yangyuoru and Hanbin Mao

Recent experiments provided controversial observations that either parallel or non-parallel G-quadruplex exists in molecularly crowded buffers that mimic cellular environment. Here, we used laser tweezers to mechanically unfold structures in a human telomeric DNA fragment, 5′-(TTAGGG)4TTA, along three different trajectories. After the end-to-end distance of each unfolding geometry was measured, it was compared with PDB structures to identify the best-matching G-quadruplex conformation. This method is well-suited to identify biomolecular structures in complex settings not amenable to conventional approaches, such as in a solution with mixed species or at physiologically significant concentrations. With this approach, we found that parallel G-quadruplex coexists with non-parallel species (1:1 ratio) in crowded buffers with dehydrating cosolutes [40% w/v dimethyl sulfoxide (DMSO) or acetonitrile (ACN)]. In crowded solutions with steric cosolutes [40% w/v bovine serum albumin (BSA)], the parallel G-quadruplex constitutes only 10% of the population. This difference unequivocally supports the notion that dehydration promotes the formation of parallel G-quadruplexes. Compared with DNA hairpins that have decreased unfolding forces in crowded (9 pN) versus diluted (15 pN) buffers, those of G-quadruplexes remain the same (20 pN). Such a result implies that in a cellular environment, DNA G-quadruplexes, instead of hairpins, can stop DNA/RNA polymerases with stall forces often <20 pN.

Friday, February 15, 2013

Flocking Multiple Microparticles with Automatically Controlled Optical Tweezers: Solutions and Experiments

Chen, H.
This paper presents an efficient approach to achieve microparticles flocking with robotics and optical tweezers technologies. All particles trapped by optical tweezers can be automatically moved toward a predefined regionwithout collision. Themain contribution of this paper lies in the proposal of several solutions to the flocking manipulation ofmicroparticles inmicroenvironments. First, a simple flocking controller is proposed to generate the desired positions and velocities for particles movement. Second, a velocity saturation method is implemented to prevent the desired velocities from exceeding a safe limit. Third, a two-layer control architecture is proposed for the motion control of optical tweezers. This architecture can help make many robotic manipulations achievable under microenvironments. The proposed approachwith these solutions can be applied to many bioapplications especially in cell engineering and biomedicine. Experiments on yeast cells with a robot-tweezers system are finally performed to verify the effectiveness of the proposed approach.

Measuring cell mechanics by optical alignment compression cytometry

Kevin B Roth , Charles Eggleton , Keith B. Neeves and David W M Marr
To address the need for a high throughput, non-destructive technique for measuring individual cell mechanical properties, we have developed optical alignment compression (OAC) cytometry. OAC combines hydrodynamic drag in an extensional flow microfluidic device with optical forces created with an inexpensive diode laser to induce measurable deformations between compressed cells. In this, a low-intensity linear optical trap aligns incoming cells with the flow stagnation point allowing hydrodynamic drag to induce deformation during cell-cell interaction. With this novel approach, we measure cell mechanical properties with a throughput that improves significantly on current non-destructive individual cell testing methods.

Formation of a metastable intramolecular RNA kissing complex by nanomanipulation

Pan T. X. Li
The stability of an RNA tertiary interaction can be affected by the region linking the two interacting domains. To examine this effect, we designed a simple RNA structure consisting of a pair of hairpins linked by a single-stranded region of 13 to 30 nucleotides. The two hairpins, each with a GACG tetraloop, can form a kissing complex of two GC base pairs. Formation of an intramolecular kissing complex extends the single-stranded linker generating an internal tension that destabilizes the tertiary interaction. Using optical tweezers, we nanomanipulated single RNA molecules into a metastable intramolecular kissing complex by offsetting the internal tension with applied force. When the kissing complex formed, the linker was stretched to nearly the full length of the kissing and hairpin helices. At such an extension, so much tension was exerted that with linkers shorter than 15 nucleotides the hairpins became unstable, which in turn prevented a kissing interaction. The RNA with a 15-nucleotide linker was able to form a transient kissing complex, an unnatural conformation, only when the applied force stretched the linker to >60% of its contour length. A 30-nucleotide linker allowed formation of a thermodynamically stable kissing complex. Experimentally observed effects of the linker length and its tension on the kissing interaction can be reasonably explained by the worm-like-chain polymer model. Our results showed the importance of internal tension and spatial separation of interacting domains in RNA tertiary folding.

Further Development of the Laser Tweezers Technique for Biomedical Applications

K Afanasiev, A Korobtsov, S Kotova, N Losevsky, A Mayorova, V Patlan and V Volostnikov
The results of the experimental work aimed at a functional enhancement of the existing laser tweezers system by introducing a pixel-addressable liquid-crystal spatial light modulator (Holoeye HEO-1080P) are presented. The use of the modulator allows us to generate light fields of complicated structure including ones with the vortex component and control the objects positions in real time. The special software is developed to form an array of optical traps with the number of elements up to thirty two with the capability of individual or subgroup control. The method of a spatial separation of the modulator aperture is implemented. The ability to control the lateral power distribution of the light field as well as the value of its orbital moment brings new possibilities of a precise manipulation of microobjects including biological ones.

Wednesday, February 13, 2013

Mammalian Myosin-18A: A Highly Divergent Myosin

Stephanie Guzik-Lendrum, Sarah M. Heissler, Neil Billington, Yasuharu Takagi, Yi Yang, Peter J. Knight, Earl Homsher and James R. Sellers

The M. musculus myosin-18A gene is expressed as two alternatively spliced isoforms, α and β, with reported roles in Golgi localization, maintenance of cytoskeleton, and as receptors for immunological surfactant proteins. Both myosin-18A isoforms feature a myosin motor domain, a single predicted IQ motif and a long coiled-coil reminiscent of myosin-2. The myosin-18Aα isoform, additionally, has an N-terminal PDZ domain. Recombinant HMM- and S1-like constructs for both myosin-18Aα and -18β species were purified from the baculovirus/Sf9 cell expression system. These constructs bound both essential and regulatory light chains indicating an additional noncanonical light chaing binding site in the neck. Myosin-18Aα and -18Aβ-S1 molecules bound actin weakly with Kd values of 4.9 and 54 uM, respectively. The actin binding data could be modeled by assuming an equilibrium between two myosin conformations, a competent and an incompetent form to bind actin. Actin binding was unchanged by presence of nucleotide. Both myosin-18A isoforms bound mant-nucleotides, but the rate of ATP hydrolysis was very slow (<0.002s-1) and not significantly enhanced by actin. Phosphorylation of the regulatory light chain had no effect on ATP hydrolysis and neither did addition of tropomyosin or of GOLPH3, a myosin-18A binding partner. Electron microscopy of myosin18A-S1 showed that the lever is strongly angled with respect to the long axis of the motor domain, suggesting a pre-powerstroke conformation regardless of the presence of ATP. These data lead us to conclude that myosin-18A does not operate as a traditional molecular motor in cells.


Electrophoretic mobility and charge inversion of a colloidal particle studied by single-colloid electrophoresis and molecular dynamics simulations

Ilya Semenov, Shervin Raafatnia, Marcello Sega, Vladimir Lobaskin, Christian Holm, and Friedrich Kremer

Optical Tweezers are employed to study the electrophoretic and the electroosmotic motion of a single colloid immersed in electrolyte solutions of ion concentrations between 10−5 and 1 mol/l and of different valencies (KCl, CaCl2, LaCl3). The measured particle mobility in monovalent salt is found to be in agreement with computations combining primitive model molecular dynamics simulations of the ionic double layer with the standard electrokinetic model. Mobility reversal of a single colloid—for the first time—is observed in the presence of trivalent ions (LaCl3) at ionic strengths larger than 10−2 mol/l. In this case, our numerical model is in a quantitative agreement with the experiment only when ion specific attractive forces are added to the primitive model, demonstrating that at low colloidal charge densities, ion correlation effects alone do not suffice to produce mobility reversal.

Orientational dynamics of human red blood cells in an optical trap

Praveen Parthasarathi; Belavadi V. Nagesh; Yogesha Lakkegowda; Shruthi S. Iyengar; Sharath Ananthamurthy; Sarbari Bhattacharya
We report here on studies of reorientation of human red blood cells (RBCs) in an optical trap. We have measured the time required, tre, for the plane of the RBC entering the optical trap to undergo a 90-deg rotation to acquire an edge on orientation with respect to the beam direction. This has been studied as a function of laser power, P, at the trap center. The variation of tre with increasing P shows an initial sharp decrease followed by a much smaller rate of further decrease. We find that this experimentally measured variation is not in complete agreement with the variation predicted by a theoretical model where the RBC is treated as a perfectly rigid circular disk-like body. We argue that this deviation arises due to deformation of the RBC. We further reason that this feature is dominated by the elastic behavior of the RBC membrane. We compare the studies carried out on normal RBCs with RBCs where varying conditions of membrane stiffness are expected. We propose that the value of energy used for maximum deformation possible during a reorientation process is an indicator of the membrane elasticity of the system under study.

Photophoretic separation of particles using two tapered optical fibers

Hongbao Xin, Dinghua Bao, Fan Zhong and Baojun Li

Particle separation is of great importance for a wide range of applications, such as biochemistry and biomedicine, and has been demonstrated using various techniques. Generally, these techniques necessitate carefully microfabricated devices and the assistance of microfluidics, thus making the separation difficult to accomplish. Here, we report a flexible, handy, and highly efficient optical method for particle separation using two tapered optical fibers, avoiding the use of complicated devices. By launching a laser beam with a power of 80 mW and wavelength of 1.55 μm into the first tapered fiber, particles of SiO2 with a size of 3.14 μm and poly(methyl methacrylate) (PMMA) with a size of 10 μm were trapped and collected. As the laser (330 mW) was switched to the second fiber for 29 s, SiO2 and PMMA particles were separated with separation efficiencies of 89.1% and 92.4%, respectively, because of their different photophoretic velocities. The separation performance was further demonstrated using different mixtures, with all separation efficiencies higher than 78%. The separation mechanism was explained by numerical simulations.

Three-dimensional optical manipulation of a single electron spin

Michael Geiselmann, Mathieu L. Juan, Jan Renger, Jana M. Say, Louise J. Brown, F. Javier García de Abajo, Frank Koppens & Romain Quidant

Nitrogen vacancy (NV) centres in diamond are promising elemental blocks for quantum optics, spin-based quantum information processing and high-resolution sensing. However, fully exploiting the capabilities of these NV centres requires suitable strategies to accurately manipulate them. Here, we use optical tweezers as a tool to achieve deterministic trapping and three-dimensional spatial manipulation of individual nanodiamonds hosting a single NV spin. Remarkably, we find that the NV axis is nearly fixed inside the trap and can be controlled in situ by adjusting the polarization of the trapping light. By combining this unique spatial and angular control with coherent manipulation of the NV spin and fluorescence lifetime measurements near an integrated photonic system, we demonstrate individual optically trapped NV centres as a novel route for both three-dimensional vectorial magnetometry and sensing of the local density of optical states.


In vivo optical trapping indicates kinesin’s stall force is reduced by dynein during intracellular transport

Benjamin H. Blehm, Trina A. Schroer, Kathleen M. Trybus, Yann R. Chemla, and Paul R. Selvin

Kinesin and dynein are fundamental components of intracellular transport, but their interactions when simultaneously present on cargos are unknown. We built an optical trap that can be calibrated in vivo during data acquisition for each individual cargo to measure forces in living cells. Comparing directional stall forces in vivo and in vitro, we found evidence that cytoplasmic dynein is active during minus- and plus-end directed motion, whereas kinesin is only active in the plus direction. In vivo, we found outward (∼plus-end) stall forces range from 2 to 7 pN, which is significantly less than the 5- to 7-pN stall force measured in vitro for single kinesin molecules. In vitro measurements on beads with kinesin-1 and dynein bound revealed a similar distribution, implying that an interaction between opposite polarity motors causes this difference. Finally, inward (∼minus-end) stalls in vivo were 2–3 pN, which is higher than the 1.1-pN stall force of a single dynein, implying multiple active dynein.


Thursday, February 7, 2013

Insights into dendritic cell function using advanced imaging modalities

Jatin M. Vyas

The application of advanced imaging techniques to fundamental questions in immunology has provided insight into dendritic cell function and has challenged dogma created using static imaging of lymphoid tissue. The history of dendritic cell biology has a storied past and is tightly linked to imaging. The development of imaging techniques that emphasize live cell imaging in situ has provided not only breath-taking movies, but also novel insights into the importance of spatiotemporal relationships between antigen presenting cells and T cells. This review serves to provide a primer on two-photon microscopy, TIRF microscopy, spinning disk confocal microscopy and optical trapping and provides selective examples of insights gained from these tools on dendritic cell biology.

Wednesday, February 6, 2013

Precise Monitoring of Chemical Changes Through Localization Analysis of Dynamic Spectra (LADS)

Smith, Zachary J.; Chang, Che-Wei; Lawson, Latevi S.; Lane, Stephen M.; Wachsmann-Hogiu, Sebastian
We present a method for monitoring subtle (sub-wavenumber) dynamics within time-varying spectra. Peak fitting is performed for large numbers of spectra in a series, allowing for monitoring time evolutions of peak positions with high precision and confidence. Sub-wavenumber peak shifts due to physical or chemical changes in the sample can be monitored and their temporal evolution characterized. In surface-enhanced Raman scattering experiments, we were able to distinguish between slow photo-damage and fast conformational change dynamics. Fluctuations in peak positions of Raman spectra recorded from a single yeast cell indicated that no significant irreversible photo-damage occurred, but these fluctuations suggest changes in the trapping conditions or biochemical changes associated with the cellular machinery in the cell. The technique is particularly suitable for applications where dynamics of spectra are of interest.

Guiding Spatial Arrangements of Ag Nanoparticles by Optical Binding Interactions in Shaped Light Fields

Zijie Yan , Raman Anand Shah , Garrett Chado ,Stephen K. Gray , Matthew Pelton , and Norbert F. Scherer
We demonstrate self-assembly of spheroidal Ag nanoparticles induced by optical binding. Particles with diameters of 40 nm formed ordered clusters and chains in aqueous solution when illuminated by shaped optical fields with a wavelength of 800 nm; specifically, close-packed clusters were formed in cylindrically symmetric optical traps, and linear chains were formed in line traps. We developed a coupled-dipole model to calculate the optical forces between an arbitrary number of particles and successfully predicted the experimentally observed particle arrangements as well as their dependence on the polarization of the incident light. This demonstrates that the interaction between these small Ag particles and light is well described by approximating the particles as point dipoles, showing that these experiments extend optical binding into the Rayleigh regime. For larger Ag nanoparticles, with diameters of approximately 100 nm, the optical-binding forces become comparable to the largest gradient forces in the optical trap, and the particles can arrange themselves into regular arrays, or synthetic photonic lattices. Finally, we discuss the differences between our experimental observations and the point dipole theory and suggest factors that prevent the Ag nanoparticles from aggregating as expected from the theory.


Measurement of Raman spectra of single airborne absorbing particles trapped by a single laser beam

Lin Ling and Yong-qing LiWe demonstrate a method for optical trapping and Raman spectroscopy of micron-sized, airborne absorbing particles using a single focused laser beam. A single Gaussian beam at 532 nm is used to trap and precisely manipulate absorbing airborne particles. The fluctuation of the position of the trapped particles is substantially reduced by controlling the power of the laser beam with a position-sensitive detector and a locking circuit. Raman spectra of the position-stabilized particles or clusters are then measured with an objective and CCD spectrograph.


Monday, February 4, 2013

Particle sorting using a subwavelength optical fiber

Yao Zhang, Baojun Li

Size-based particle sorting using a subwavelength optical fiber was demonstrated with 600-nm and 1-μm sizes of polystyrene particles. Optical forces acting on the particles were calculated based on three-dimensional finite-difference time-domain simulations at wavelengths of 808, 1047, and 1310 nm propagating in a subwavelength optical fiber with diameter of 800 nm. Calculations indicate that by launching two counterpropagating laser beams at different wavelengths into the fiber, the directions of the resultant optical scattering forces acting on the two particle sizes can be opposite along the fiber, which leads to a countertransport of the particles. To verify the theoretical prediction, experiments were performed using the 800-nm fiber to sort the two particle sizes. The results show that with two counterpropagating beams at 808 and 1310 nm, a continuous particle sorting was achieved. Measured particle velocities were in agreement with the theoretical calculations.

Effect of handle length and microsphere size on transition kinetics in single-molecule experiments

Jen-Chien Chang, Michel de Messieres, and Arthur La Porta
When subject to constant tension, a DNA or RNA hairpin will typically make abrupt transitions between the open and closed state. Although the transition kinetics are an intrinsic property of the molecule, the transition rates measured in single-molecule experiments can be influenced by the configuration of the measurement system. We investigate the transition kinetics for a DNA hairpin held under constant force by an optical trap as a function of microsphere size and double-stranded DNA handle length. We find the apparent transition lifetime cannot be expressed as a function of the drag coefficient of the microsphere alone or as a function of time scales relevant to the optical trap. The apparent transition lifetime is found to be a linear function of the factor βeff·αhandle, where βeff is the effective drag coefficient of the microsphere near the surface and αhandle is the stiffness of the DNA tether. The results provide insight into the perturbation to the hairpin transition kinetics due to experimental configuration and guidance for designing single-molecule experiments which determine the intrinsic molecular kinetics.

Friday, February 1, 2013

From infection to detection: imaging S. aureus--host interactions

U. Neugebauer , C. Grosse , M. Bauer , B. Kemper , A. Barroso-Pena and A. Bauwens
Infections, particularly those due to drug-resistant pathogens, significantly increase morbidity and mortality as well as cost of treatment and length of hospital stays. Staphylococcus aureus, a highly human-adapted organism, is the most common pathogen causing nosocomial infections. Among S. aureus, especially methicillin-resistant S. aureus (MRSA) causes problems in therapy and infection control. Understanding the mechanisms of infections is as important as the development of rapid tools for diagnosis. Within the Photonics4Life project "From Infection to Detection" these two goals are addressed. Modern optical technologies, such as multi-focus quantitative digital holographic microscopy (DHM) phase contrast, holographic optical tweezers (HOT) and Raman spectroscopy have been employed to analyse the cell morphology, cell dynamics and cellular refractive index of endothelial cells before and after incubation with S. aureus (or with model bacteria for HOT and DHM analysis). Individual bacteria inside the cells have been visualized and defined infection scenarios at the single cell level could be created. Finally, optical techniques were evaluated for further sub-typing of S. aureus strains and compared to the established spa typing method.


Highly Controllable Optical Tweezers Using Dynamic Electronic Holograms

Yamamoto, Johtaro; Iwai, Toshiaki
Dielectric particles including living cells are trapped within focused laser beam spots, and as a result, they can be transferred by displacing the beam spots. Such the particle manipulating technique is called optical tweezers. Holographic optical tweezers (HOT) enables highly flexible and precise control of particles, introducing holography technique to them. HOT is one of the most expected techniques for investigations of cell-cell signaling which require precise arraying of living cells. We had developed a new highly controllable HOT system where two different intensity patterns, a carrier beam spot and a beam array, are generated quasi-simultaneously by time-division multiplexing. Particles are transferred to the beam array by the carrier beam spot displaced in real time by phase shifting of holograms. In this review, we introduce our work, the construction of the system, demonstration of manipulating particles and investigations of the spatio- temporal stability of trapped particles in our system.

Serial Raman spectroscopy of particles trapped on a waveguide

Pål Løvhaugen, Balpreet Singh Ahluwalia, Thomas R. Huser, and Olav Gaute Hellesø

We demonstrate that Raman spectroscopy can be used to characterize and identify particles that are trapped and propelled along optical waveguides. To accomplish this, microscopic particles on a waveguide are moved along the waveguide and then individually addressed by a focused laser beam to obtain their characteristic Raman signature within 1 second acquisition time. The spectrum is used to distinguish between glass and polystyrene particles. After the characterization, the particles continue to be propelled along the straight waveguide. Alternatively, a waveguide loop with a gap is also investigated, and in this case particles are held in the gap for characterization before they are released.


Optical tweezers with fluorescence detection for temperature-dependent microrheological measurements

Atsuomi Shundo, Koichiro Hori, David P. Penaloza, Jr., and Keiji Tanaka

We introduce a setup of optical tweezers, capable of carrying out temperature-dependent rheological measurements of soft materials. In our setup, the particle displacement is detected by imaging a bright spot due to fluorescence emitted from a dye-labeled particle against a dark background onto a quadrant photodiode. This setup has a relatively wide space around the sample that allows us to further accessorize the optical tweezers by a temperature control unit. The applicability of the setup was examined on the basis of the rheological measurements using a typical viscoelastic system, namely a worm-like micelle solution. The temperature and frequency dependences of the local viscoelastic functions of the worm-like micelle solution obtained by this setup were in good accordance with those obtained by a conventional oscillatory rheometer, confirming the capability of the optical tweezers as a tool for the local rheological measurements of soft materials. Since the optical tweezers measurements only require a tiny amount of sample (∼40 μL), the rheological measurements using our setup should be useful for soft materials of which the available amount is limited.

Photocatalytic nano-optical trapping using TiO2 nanosphere pairs mediated with Mie-scattered near field

Toshiyuki Honda, Mitsuhiro Terakawa, Minoru Obara
The localized enhanced near field on nanostructures has been attracting much attention for a template for size-selective optical trapping (tweezers) beyond the diffraction limit. The near-field optical trapping has mainly been studied using metallic substrates such as Au nanodot pairs, periodic Al nanoslits, nanoapertures on an Au film, etc. In this paper, we newly propose a Mie-scattered-near-field optical trapping scheme for size-selective photocatalytic application using pairs of poly-rutile TiO2 nanospheres. The optical intensity distribution in a 3D-nanogap space between the nanospheres was simulated by a 3D FDTD method. The simulation system consists of the two TiO2nanospheres placed on a silica substrate in water. The 400-nm excitation laser is used for both the near-field trapping and the photocatalyst excitation. The optical trapping forces were calculated based on the near-field optical intensity distribution. The trapping stiffness for 20-nm polystyrene sphere at a gap distance of 20 nm was 6.4 pN/nm/W. The optical force vector shows that the object like virus can be trapped with sufficient forces into the nanogap space and then is driven into the direct surface of the TiO2 sphere. This result suggests that this system works as a photocatalytic trapping for killing virus, protein, etc.

Force Mapping during the Formation and Maturation of Cell Adhesion Sites with Multiple Optical Tweezers

Melanie Schwingel, Martin Bastmeyer

Focal contacts act as mechanosensors allowing cells to respond to their biomechanical environment. Force transmission through newly formed contact sites is a highly dynamic process requiring a stable link between the intracellular cytoskeleton and the extracellular environment. To simultaneously investigate cellular traction forces in several individual maturing adhesion sites within the same cell, we established a custom-built multiple trap optical tweezers setup. Beads functionalized with fibronectin or RGD-peptides were placed onto the apical surface of a cell and trapped with a maximum force of 160 pN. Cells form adhesion contacts around the beads as demonstrated by vinculin accumulation and start to apply traction forces after 30 seconds. Force transmission was found to strongly depend on bead size, surface density of integrin ligands and bead location on the cell surface. Highest traction forces were measured for beads positioned on the leading edge. For mouse embryonic fibroblasts, traction forces acting on single beads are in the range of 80 pN after 5 minutes. If two beads were positioned parallel to the leading edge and with a center-to-center distance less than 10 µm, traction forces acting on single beads were reduced by 40%. This indicates a spatial and temporal coordination of force development in closely related adhesion sites. We also used our setup to compare traction forces, retrograde transport velocities, and migration velocities between two cell lines (mouse melanoma and fibroblasts) and primary chick fibroblasts. We find that maximal force development differs considerably between the three cell types with the primary cells being the strongest. In addition, we observe a linear relation between force and retrograde transport velocity: a high retrograde transport velocity is associated with strong cellular traction forces. In contrast, migration velocity is inversely related to traction forces and retrograde transport velocity.


Manipulating Cell Adhesions with Optical Tweezers for Study of Cell-to-Cell Interactions

Hu, Songyu; Gou, Xue; Han, Hochun; Leung, Anskar Y.H.; Sun, Dong
This paper presents an approach to manipulating cell adhesions using optical tweezers for cell-to-cell interactions at single cell level. A case study of investigating the adhesions between leukemia cells and bone marrow stromal cells is reported. First, the trapping force imposed on the cell is calibrated and the viability of leukemia cells after optical trapping is tested and verified. This is for demonstrating the feasibility of the proposed optical manipulation method. Second, properties of adhesions of leukemia cells K562 on stromal cells M210B4 from mouse and HS5 from human are characterized. Based on characterization results, we classify adhesions into three categories namely tightly adherent, loosely adherent or free suspending. Finally, the adhesion abilities of K562 on M210B4 and HS5 are changed by adding heparin into culture medium, which demonstrates the specificity of the adhesion. The important contribution of this paper lies in development of a dexterous cell manipulation method to characterize cell adhesion properties, which helps create a new opportunity to investigate cell-to-cell interactions at single cell level.