Simulation of the acceleration mechanism by light-propulsion for the powder particles at laser direct material deposition

I.О. Kovaleva, O.B. Kovalev

The results of the numerical analysis of heat- and mass-transfer processes at powder particles' motion in a gas flow and laser beam by light-propulsion force during the laser cladding and direct material deposition are presented. Under consideration were the stainless steel particles, the radiation power range of the CO2 laser were 1000, 3000 and 5000 W. Finally, the particles of 45 μm in diameter reach the maximum velocity of about 80, 220, 280 m/s. It is shown that as particles are heated by the laser up to the temperature approaching the boiling point, the particles' velocity in the light field by the vapor recoil pressure may increase significantly. The radius of the particles slightly varies due to the evaporation; the losses in the clad material mass are negligibly small. Comparisons of numerical results with known experimental data on light-propulsion acceleration of single particles (aluminum, aluminum oxide and graphite) under the influence of pulse laser radiation are also presented. Particle acceleration resulting from the laser evaporation depends on the particle diameter, powder material properties, focusing degree and attenuation laser beam intensity by the direction of its propagation.

DOI

Optical trapping: A review of essential concepts

I.Verdeny, A.Farré, J.Mas, C.López-Quesada, E.Martín-Badosa, M.Montes-Usategui

Optical tweezers are an innovative technique for the non-contact, all-optical manipulation of small material samples, which has extraordinarily expanded and evolved since its inception in the mid-80s of the last century. Nowadays, the potential of optical tweezers has been clearly proven and a wide range of applications both from the physical and biological sciences have solidly emerged, turning the early ideas and techniques into a powerful paradigm for experimentation in the micro- and nanoworld. This review aims at highlighting the fundamental concepts that are essential for a thorough understanding of optical trapping, making emphasis on both its manipulation and measurement capabilities, as well as on the vast array of important biological applications appeared in the last years.

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Multimodal biophotonic workstation for live cell analysis

Michael Esseling, Björn Kemper, Maciej Antkowiak, David J. Stevenson, Lionel Chaudet, Mark A. A. Neil, Paul W. French, Gert von Bally, Kishan Dholakia, Cornelia Denz

A reliable description and quantification of the complex physiology and reactions of living cells requires a multimodal analysis with various measurement techniques. We have investigated the integration of different techniques into a biophotonic workstation that can provide biological researchers with these capabilities. The combination of a micromanipulation tool with three different imaging principles is accomplished in a single inverted microscope which makes the results from all the techniques directly comparable. Chinese Hamster Ovary (CHO) cells were manipulated by optical tweezers while the feedback was directly analyzed by fluorescence lifetime imaging, digital holographic microscopy and dynamic phase-contrast microscopy.

DOI

Microfabricated continuous cubic phase plate induced Airy beams for optical manipulation with high power efficiency

Rui Cao, Yong Yang, Jingang Wang, Jing Bu, Mingwei Wang, and X.-C. Yuan

We studied and demonstrated optical trapping capabilities of an Airy beam generated with a cubic phase plate incorporated into a conventional optical tweezer system. The power efficiency and damage threshold of the cubic phase plate were found to be much higher when spatial light modulators were employed in beam generation.

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A multi-mode fiber probe for holographic micromanipulation and microscopy

Silvio Bianchi and Roberto Di Leonardo

Holographic tweezers have revolutionized the way we do experiments at the micron scale. The possibility of applying controlled force fields on simultaneously trapped micro-particles has allowed to directly probe interactions and mechanical properties of colloids, macromolecules and living cells. Holographic micromanipulation requires the careful shaping of a laser beam that is then focused by a microscope objective onto a micro-hologram in the sample volume. The same objective is used for imaging. That approach is therefore limited to in vitro samples contained in transparent cells that are easily accessed optically. Here we demonstrate that the complex light propagator of a real multimode fiber can be directly measured. That allows to transmit a micro-hologram through a 1 metre long (60 μm core) optical fiber and produce dynamic arrays of focused spots at the fiber output. We show that those spots can be used for interactive holographic micromanipulation of micron sized beads facing the fiber tip. Scanning a single spot across the output fiber we can employ the same fiber as a probe for scanning fluorescence microscopy. Our findings open the way towards the fabrication of endoscopic probes which could be capable of seeing and manipulating single cells deep into biological tissues.

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Metrology of laser-guided particles in air-filled hollow-core photonic crystal fiber

O. A. Schmidt, M. K. Garbos, T. G. Euser, and P. St. J. Russell

Micrometer-sized particles are trapped in front of an air-filled hollow-core photonic crystal fiber using a novel dual-beam trap. A backward guided mode produces a divergent beam that diffracts out of the core, and simultaneously a focused laser beam launches a forward-propagating mode into the core. By changing the backward/forward power balance, a trapped particle can be selectively launched into the hollow core. Once inside, particles can be optically propelled along several meters of fiber with mobilities as high as 19  cm·s−1  W−1 (precisely measured using in-fiber Doppler velocimetry). The results are in excellent agreement with theory. The system allows determination of fiber loss as well as the mass density and refractive index of single particles.

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Large-area optoelastic manipulation of colloidal particles in liquid crystals using photoresponsive molecular surface monolayers

Angel Martinez, Hector C. Mireles, and Ivan I. Smalyukh

Noncontact optical trapping and manipulation of micrometer- and nanometer-sized particles are typically achieved by use of forces and torques exerted by tightly focused high-intensity laser beams. Although they were instrumental for many scientific breakthroughs, these approaches find few technological applications mainly because of the small-area manipulation capabilities, the need for using high laser powers, limited application to anisotropic fluids and low-refractive-index particles, as well as complexity of implementation. To overcome these limitations, recent research efforts have been directed toward extending the scope of noncontact optical control through the use of optically-guided electrokinetic forces, vortex laser beams, plasmonics, and optofluidics. Here we demonstrate manipulation of colloidal particles and self-assembled structures in nematic liquid crystals by means of single-molecule-thick, light-controlled surface monolayers. Using polarized light of intensity from 1,000 to 100,000 times smaller than that in conventional optical tweezers, we rotate, translate, localize, and assemble spherical and complex-shaped particles of various sizes and compositions. By controlling boundary conditions through the monolayer, we manipulate the liquid crystal director field and the landscape of ensuing elastic forces exerted on colloids by the host medium. This permits the centimeter-scale, massively parallel manipulation of particles and complex colloidal structures that can be dynamically controlled by changing illumination or assembled into stationary stable configurations dictated by the “memorized” optoelastic potential landscape due to the last illumination pattern. We characterize the strength of optically guided elastic forces and discuss the potential uses of this noncontact manipulation in fabrication of novel optically- and electrically-tunable composites from liquid crystals and colloids.

DOI

Realization of a micrometre-sized stochastic heat engine

Valentin Blickle & Clemens Bechinger

The conversion of energy into mechanical work is essential for almost any industrial process. The original description of classical heat engines by Sadi Carnot in 1824 has largely shaped our understanding of work and heat exchange during macroscopic thermodynamic processes. Equipped with our present-day ability to design and control mechanical devices at micro- and nanometre length scales, we are now in a position to explore the limitations of classical thermodynamics, arising on scales for which thermal fluctuations are important. Here we demonstrate the experimental realization of a microscopic heat engine, comprising a single colloidal particle subject to a time-dependent optical laser trap. The work associated with the system is a fluctuating quantity, and depends strongly on the cycle duration time, τ, which in turn determines the efficiency of our heat engine. Our experiments offer a rare insight into the conversion of thermal to mechanical energy on a microscopic level, and pave the way for the design of future micromechanical machines.

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Mutations Altering the Interplay between GkDnaC Helicase and DNA Reveal an Insight into Helicase Unwinding

Yu-Hua Lo, Shih-Wei Liu, Yuh-Ju Sun, Hung-Wen Li,Chwan-Deng Hsiao

Replicative helicases are essential molecular machines that utilize energy derived from NTP hydrolysis to move along nucleic acids and to unwind double-stranded DNA (dsDNA). Our earlier crystal structure of the hexameric helicase from Geobacillus kaustophilus HTA426 (GkDnaC) in complex with single-stranded DNA (ssDNA) suggested several key residues responsible for DNA binding that likely play a role in DNA translocation during the unwinding process. Here, we demonstrated that the unwinding activities of mutants with substitutions at these key residues inGkDnaC are 2–4-fold higher than that of wild-type protein. We also observed the faster unwinding velocities in these mutants using single-molecule experiments. A partial loss in the interaction of helicase with ssDNA leads to an enhancement in helicase efficiency, while their ATPase activities remain unchanged. In strong contrast, adding accessory proteins (DnaG or DnaI) to GkDnaC helicase alters the ATPase, unwinding efficiency and the unwinding velocity of the helicase. It suggests that the unwinding velocity of helicase could be modulated by two different pathways, the efficiency of ATP hydrolysis or protein-DNA interaction.

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Low-power nano-optical vortex trapping via plasmonic diabolo nanoantennas

Ju-Hyung Kang, Kipom Kim, Ho-Seok Ee, Yong-Hee Lee, Tae-Young Yoon, Min-Kyo Seo & Hong-Gyu Park

Optical vortex trapping can allow the capture and manipulation of micro- and nanometre-sized objects such as damageable biological particles or particles with a refractive index lower than the surrounding material. However, the quest for nanometric optical vortex trapping that overcomes the diffraction limit remains. Here we demonstrate the first experimental implementation of low-power nano-optical vortex trapping using plasmonic resonance in gold diabolo nanoantennas. The vortex trapping potential was formed with a minimum at 170 nm from the central local maximum, and allowed polystyrene nanoparticles in water to be trapped strongly at the boundary of the nanoantenna. Furthermore, a large radial trapping stiffness, ~0.69 pN nm−1W−1, was measured at the position of the minimum potential, showing good agreement with numerical simulations. This subwavelength-scale nanoantenna system capable of low-power trapping represents a significant step toward versatile, efficient nano-optical manipulations in lab-on-a-chip devices.

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Three-dimensional imaging and force characterization of multiple trapped particles in low NA counterpropagating optical traps

T. B. Lindballe, M. V. Kristensen, A. P. Kylling, D. Z. Palima, J. Glückstad, S. R. Keiding, H. Stapelfeldt

An experimental characterization of the three-dimensional (3D) position and force constants, acting on one or multiple trapped polystyrene beads in a weak counterpropagating beams geometry is reported. The 3D position of the trapped particles is tracked by imaging with two synchronized CMOS cameras from two orthogonal views and used to determine the stiffness along all three spatial directions through power spectrum analysis and the equipartition method. For the case of three trapped beads we measure the dependence of the force constants on the counterpropagating beams waist separation. The maximal transverse stiffnesses, is about 0.1 pN/µm per mW at a beam waist separation of 67 µm whereas the longitudinal stiffness is approximately 20 times lower. The experimental findings are in reasonable agreement with a recent physical-geometric optics calculation.

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Optical Trapping of a Single Protein

Yuanjie Pang and Reuven Gordon

We experimentally demonstrate the optical trapping of a single bovine serum albumin (BSA) molecule that has a hydrodynamic radius of 3.4 nm, using a double-nanohole in an Au film. The strong optical force in the trap not only stably traps the protein molecule but also unfolds it. The unfolding of the BSA is confirmed by experiments with changing optical power and with changing solution pH. The detection of the trapping event has a signal-to-noise ratio of 33, which shows that the setup is extremely sensitive to detect the presence of a protein, even at the single molecule level.

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Advances in Experiments and Modeling in Micro- and Nano-Biomechanics: A Mini Review

Mian Long, Masaaki Sato, Chwee Teck Lim, Jianhua Wu, Taiji Adachiand Yasuhiro Inoue

Recent advances in micro- and nano-technologies and high-end computing have enabled the development of new experimental and modeling approaches to study biomechanics at the micro- and nano-scales that were previously not possible. These new cutting-edge approaches are contributing toward our understanding in emerging areas such as mechanobiology and mechanochemistry. Another important potential contribution lies in translational medicine, since biomechanical studies at the cellular and molecular levels have direct relevance in areas such disease diagnosis, nano-medicine and drug delivery. Thus, the developed experimental and modeling approaches are critical in elucidating important mechanistic insights in both basic sciences and clinical treatment. While it is hard to cover all the recent advances in this mini-review, we focus on several important approaches. For experimental techniques, we review the assays involving shear flow, cellular imaging, microbead, microcontact printing, and micropillars at the micro-scale, and micropipette aspiration, optical tweezers, parallel flow chamber, and atomic force microscopy at the nano-scale. In modeling and simulations, we outline the theoretical modeling for actin dynamics in migrating cell and actin-based cell motility in cellular mechanics, as well as the receptor–ligand binding in cell adhesion and the application of free, steered, and flow molecular dynamics simulations in molecular biomechanics. Relevant scientific issues and applications are also discussed.

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Progress in coagulation rate measurements of colloidal dispersions

Shenghua Xu and Zhiwei Sun

This article reviews recent advances in coagulation rate measurements of colloidal dispersions, with emphasis on the turbidity method. For turbidity method, measurement of the coagulation rate relies upon the turbidity change resulting from the coagulation process, and the measuring sensitivity significantly depends on particle size and the wavelength used. There exists a “zero sensitivity” blind point for measurement at a specific wavelength, suggesting that such measurements should be performed at a wavelength some distance from the blind point. The major difficulty in determining absolute coagulation rate constant (CRC) by light scattering and turbidity measurements is how to theoretically solve the scattering problem of 2-particle aggregates. The T-matrix method accurately solves this problem, showing its superiority over various earlier theoretical approximations (applicable only to small particles). Results from studies on effects of forward scattering, multiple scattering, etc., provide guidelines for choosing proper particle size and volume fraction for the allowed margin of measurement error. Most of these findings on turbidity methods are also valid or applicable to other scattering methods. Finally, we introduce a new microscopic approach to assess the colloidal stability at individual particle levels, by means of directly observing artificially induced collision with the aid of optical tweezers.

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Optical trapping for analytical biotechnology

Praveen C Ashok, Kishan Dholakia

We describe the exciting advances of using optical trapping in the field of analytical biotechnology. This technique has opened up opportunities to manipulate biological particles at the single cell or even at subcellular levels which has allowed an insight into the physical and chemical mechanisms of many biological processes. The ability of this technique to manipulate microparticles and measure pico-Newton forces has found several applications such as understanding the dynamics of biological macromolecules, cell–cell interactions and the micro-rheology of both cells and fluids. Furthermore we may probe and analyse the biological world when combining trapping with analytical techniques such as Raman spectroscopy and imaging.

DOI

Quasiperiodic Distribution of Rigor Cross-Bridges Along a Reconstituted Thin Filament in a Skeletal Myofibril

Madoka Suzuki, Shin'ichi Ishiwata

Electron microscopy has shown that cross-bridges (CBs) are formed at the target zone that is periodically distributed on the thin filament in striated muscle. Here, by manipulating a single bead-tailed actin filament with optical tweezers, we measured the unbinding events of rigor CBs one by one on the surface of the A-band in rabbit skeletal myofibrils. We found that the spacings between adjacent CBs were not always the same, and instead were 36, 72, or 108 nm. Tropomyosin and troponin did not affect the CB spacing except for a relative increase in the appearance of longer spacing in the presence of Ca2+. In addition, in an in vitro assay where myosin molecules were randomly distributed, were obtained the same spacing, i.e., a multiple of 36 nm. These results indicate that the one-dimensional distribution of CBs matches with the 36-nm half pitch of a long helical structure of actin filaments. A stereospecific model composed of three actin protomers per target zone was shown to explain the experimental results. Additionally, the unbinding force (i.e., the binding affinity) of CBs for the reconstituted thin filaments was found to be larger and smaller relative to that for actin filaments with and without Ca2+, respectively.

DOI

Cooperative Responses of Multiple Kinesins to Variable and Constant Loads

D. Kenneth Jamison, Jonathan W. Driver and Michael R. Diehl

Microtubule-dependent transport is most often driven by collections of kinesins and dyneins that function in either a concerted fa-shion or antagonistically. Several lines of evi-dence suggest that cargo transport may not be influenced appreciably by the combined action of multiple kinesins. Yet, as in previous optical trapping experiments, the forces imposed on cargos will vary spatially and temporally in cells depending on a number of local environ-mental factors, and the influence of these con-ditions has been largely overlooked. Here, we characterize the dynamics of structurally-defined complexes containing multiple kinesins under controlled loads of an optical force clamp. While demonstrating that there are ge-neric kinetic barriers that restrict the ability of multiple kinesins to cooperate productively, the spatial and temporal properties of applied loads is found to play an important role in the collective dynamics of multiple-motor systems. We propose these dependencies have implica-tions for intracellular transport processes, es-pecially for bidirectional transport.

DOI

Optimisation of a low cost SLM for diffraction efficiency and ghost order suppression

R. Bowman, V. D’Ambrosio, E. Rubino, O. Jedrkiewicz, P. Di Trapani and M. J. Padgett

Spatial Light Modulators (SLMs) are a powerful tool in many optics laboratories, but due to the technology required for their fabrication, they are usually very expensive. Recently some inexpensive devices have been produced, however their phase shift range is less than 2π, leading to a loss of diffraction efficiency for the SLM. We show how to improve the first order diffraction efficiency of such an SLM by adjusting the blazing function, and obtain a 1.5 times increase in first order diffracted power. Even a perfect SLM with 2π phase throw can produce undesired effects in some situations; for example in holographic optical tweezers it is common to find unwanted “ghost spots” near to the array of first-order spots. Modulating the amplitude, by spatially modulating the blazing function, allows us to suppress the ghost spots. This increases the contrast between desired and unwanted spots by more than an order of magnitude.

DOI

A photon-driven micromotor can direct nerve fibre growth

Tao Wu, Timo A. Nieminen, Samarendra Mohanty, Jill Miotke, Ronald L. Meyer, Halina Rubinsztein-Dunlop & Michael W. Berns

Axonal path-finding is important in the development of the nervous system, nerve repair and nerve regeneration. The behaviour of the growth cone at the tip of the growing axon determines the direction of axonal growth and migration. We have developed an optical-based system to control the direction of growth of individual axons (nerve fibres) using laser-driven spinning birefringent spheres. One or two optical traps position birefringent beads adjacent to growth cones of cultured goldfish retinal ganglion cell axons. Circularly polarized light with angular momentum causes the trapped bead to spin. This creates a localized microfluidic flow generating an estimated 0.17 pN shear force against the growth cone that turns in response to the shear. The direction of axonal growth can be precisely manipulated by changing the rotation direction and position of this optically driven micromotor. A physical model estimating the shear force density on the axon is described.

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Non-specific interactions of CdTe/Cds Quantum Dots with human blood mononuclear cells

Rafael B. Lira, Mariana B. Cavalcanti, Maria A.B.L. Seabra, Diego C.N. Silva, Ademir J. Amaral, Beate S. Santos, Adriana Fontes

In order to study biological events, researchers commonly use methods based on fluorescence. These techniques generally use fluorescent probes, commonly small organic molecules or fluorescent proteins. However, these probes still present some drawbacks, limiting the detection. Semiconductor nanocrystals – Quantum Dots (QDs) – have emerged as an alternative tool to conventional fluorescent dyes in biological detection due to its topping properties – wide absorption cross section, brightness and high photostability. Some questions have emerged about the use of QDs for biological applications. Here, we use optical tools to study non-specific interactions between aqueous synthesized QDs and peripheral blood mononuclear cells. By fluorescence microscopy we observed that bare QDs can label cell membrane in live cells and also label intracellular compartments in artificially permeabilized cells, indicating that non-specific labeling of sub-structures inside the cells must be considered when investigating an internal target by specific conjugation. Since fluorescence microscopy and flow cytometry are complementary techniques (fluorescence microscopy provides a morphological image of a few samples and flow cytometry is a powerful technique to quantify biological events in a large number of cells), in this work we also used flow cytometry to investigate non-specific labeling. Moreover, by using optical tweezers, we observed that, after QDs incubation, zeta potentials in live cells changed to a less negative value, which may indicate that oxidative adverse effects were caused by QDs to the cells.

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The formation of actin waves during regeneration after axonal lesion is enhanced by BDNF

Francesco Difato, Hanako Tsushima, Mattia Pesce, Fabio Benfenati, Axel Blau & Evelina Chieregatti

During development, axons of neurons in the mammalian central nervous system lose their ability to regenerate. To study the regeneration process, axons of mouse hippocampal neurons were partially damaged by an UVA laser dissector system. The possibility to deliver very low average power to the sample reduced the collateral thermal damage and allowed studying axonal regeneration of mouse neurons during early days in vitro. Force spectroscopy measurements were performed during and after axon ablation with a bead attached to the axonal membrane and held in an optical trap. With this approach, we quantified the adhesion of the axon to the substrate and the viscoelastic properties of the membrane during regeneration. The reorganization and regeneration of the axon was documented by long-term live imaging. Here we demonstrate that BDNF regulates neuronal adhesion and favors the formation of actin waves during regeneration after axonal lesion.

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Single cell Raman spectroscopy for cell sorting and imaging

Mengqiu Li, Jian Xu, Maria Romero-Gonzalez, Steve A Banwart, Wei E Huang

Single cell Raman spectroscopy (SCRS) is a non-invasive and label-free technology, allowing in vivo and multiple parameter analysis of individual living cells. A single cell Raman spectrum usually contains more than 1000 Raman bands which provide rich and intrinsic information of the cell (e.g. nucleic acids, protein, carbohydrates and lipids), reflecting cellular genotypes, phenotypes and physiological states. A Raman spectrum serves as a molecular ‘fingerprint’ of a single cell, making it possible to differentiate various cells including bacterial, protistan and animal cells without prior knowledge of the cells. However, a key drawback of SCRS is the fact that spontaneous Raman signals are naturally weak; this review discusses recent research progress in significantly enhancing and improving the signal of spontaneous Raman spectroscopy, including resonance Raman spectroscopy (RRS), coherent anti-Stokes Raman spectroscopy (CARS), stimulated Raman spectroscopy (SRS) and surface enhanced Raman scattering (SERS). This review focuses on the biotechnological development and the associated applications of SCRS, including Raman activated cell sorting (RACS) and Raman imaging and mapping.

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Phase-Transition-like Properties of Double-Beam Optical Tweezers

A. B. Stilgoe, N. R. Heckenberg, T. A. Nieminen, and H. Rubinsztein-Dunlop

We report on double-beam optical tweezers that undergo previously unknown phase-transition-like behavior resulting in the formation of more optical traps than the number of beams used to create them. We classify the optical force fields which produce multiple traps for a double-beam system including the critical behavior. This effect is demonstrated experimentally in orthogonally polarized (noninterfering) dual-beam optical tweezers for a silica particle of 2.32  μm diameter. Phase transitions of multiple beam trapping systems have implications for hopping rates between traps and detection of forces between biomolecules using dual-beam optical tweezers. It is an example of a novel dynamic system with multiple states where force fields undergo a series of sign inversions as a function of parameters such as size and beam separation.

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Extending vaterite microviscometry to ex vivo blood vessels by serial calibration

Samir G. Shreim, Earl Steward, and Elliot L. Botvinick

The endothelial glycocalyx layer is a ~2 µm thick glycosaminoglycan rich pericellular matrix expressed on the luminal surface of vascular endothelial cells, which has implications in vessel mechanics and mechanotransduction. Despite its role in vascular physiology, no direct measurement has of yet been made of vessel glycocalyx material properties. Vaterite microviscometry is a laser tweezers based microrheological method, which has been previously utilized to measure the viscosity of linear and complex fluids under flow. This form of microrheology has until now relied on complete recollection of the forward scattered light. Here we present a novel method to extend vaterite microviscometry to relatively thick samples. We validate our method and its assumptions and measure the apparent viscosity as a function of distance from the vascular endothelium. We observe a differential response in conditions designed to preserve the EGL in comparison to those designed to collapse it.

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Actin filaments function as a tension sensor by tension-dependent binding of cofilin to the filament

Kimihide Hayakawa, Hitoshi Tatsumi, and Masahiro Sokabe

Intracellular and extracellular mechanical forces affect the structure and dynamics of the actin cytoskeleton. However, the underlying molecular and biophysical mechanisms, including how mechanical forces are sensed, are largely unknown. Actin-depolymerizing factor/cofilin proteins are actin-modulating proteins that are ubiquitously distributed in eukaryotes, and they are the most likely candidate as proteins to drive stress fiber disassembly in response to changes in tension in the fiber. In this study, we propose a novel hypothesis that tension in an actin filament prevents the filament from being severed by cofilin. To test this, we placed single actin filaments under tension using optical tweezers. When a fiber was tensed, it was severed after the application of cofilin with a significantly larger delay in comparison with control filaments suspended in solution. The binding rate of cofilin to an actin bundle decreased when the bundle was tensed. These results suggest that tension in an actin filament reduces the cofilin binding, resulting in a decrease in its effective severing activity.

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Bimolecular integrin–ligand interactions quantified using peptide-functionalized dextran-coated microparticles

Jessie E. P. Sun, Justin Vranic, Russell J. Composto, Craig Streu, Paul C. Billings, Joel S. Bennett, John W. Weisel and Rustem I. Litvinov
Integrins play a key role in cell–cell and cell–matrix interactions. Artificial surfaces grafted with integrin ligands, mimicking natural interfaces, have been used to study integrin-mediated cell adhesion. Here we report the use of a new chemical engineering technology in combination with single-molecule nanomechanical measurements to quantify peptide binding to integrins. We prepared latex beads with covalently-attached dextran. The beads were then functionalized with the bioactive peptides, cyclic RGDFK (cRGD) and the fibrinogen γC-dodecapeptide (H12), corresponding to the active sites for fibrinogen binding to the platelet integrin αIIbβ3. Using optical tweezers-based force spectroscopy to measure non-specific protein–protein interactions, we found the dextran-coated beads nonreactive towards fibrinogen, thus providing an inert platform for biospecific modifications. Using periodate oxidation followed by reductive amination, we functionalized the bead-attached dextran with either cRGD or H12 and used the peptide-grafted beads to measure single-molecule interactions with the purified αIIbβ3. Bimolecular force spectroscopy revealed that the peptide-functionalized beads were highly and specifically reactive with the immobilized αIIbβ3. Further, the cRGD- and H12-functionalized beads displayed a remarkable interaction profile with a bimodal force distribution up to 90 pN. The cRGD–αIIbβ3 interactions had greater binding strength than that of H12–αIIbβ3, indicating that they are more stable and resistant mechanically, consistent with the platelet reactivity of RGD-containing ligands. Thus, the results reported here describe the mechanistic characteristics of αIIbβ3–ligand interactions, confirming the utility of peptide-functionalized latex beads for the quantitative analysis of molecular recognition.

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Enhanced Optical Trapping and Arrangement of Nano-Objects in a Plasmonic Nanocavity

Chang Chen, Mathieu L. Juan, Yi Li, Guido Maes, Gustaaf Borghs, Pol Van Dorpe, and Romain Quidant

Gentle manipulation of micrometer-sized dielectric objects with optical forces has found many applications in both life and physical sciences. To further extend optical trapping toward the true nanometer scale, we present an original approach combining self-induced back action (SIBA) trapping with the latest advances in nanoscale plasmon engineering. The designed resonant trap, formed by a rectangular plasmonic nanopore, is successfully tested on 22 nm polystyrene beads, showing both single- and double-bead trapping events. The mechanism responsible for the higher stability of the double-bead trapping is discussed, in light of the statistical analysis of the experimental data and numerical calculations. Furthermore, we propose a figure of merit that we use to quantify the achieved trapping efficiency and compare it to prior optical nanotweezers. Our approach may open new routes toward ultra-accurate immobilization and arrangement of nanoscale objects, such as biomolecules.

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Dark-field optical tweezers for nanometrology of metallic nanoparticles

Kellie Pearce, Fan Wang, and Peter J. Reece

Applications of metallic nanoparticles are based on their strongly size-dependent optical properties. We present a method for combining optical tweezers with dark field microscopy that allows measurement of localised surface plasmon resonance (LSPR) spectra on single isolated nanoparticles without compromising the strength of the optical trap. Using this spectroscopic information in combination with measurements of trap stiffness and hydrodynamic drag, allows us to determine the dimensions of the trapped nanoparticles. A relationship is found between the measured diameters of the particles and the peak wavelengths of their spectra. Using this method we may also resolve complex spectra of particle aggregation and interactions within the tweezers.

DOI

Probing ribosomal protein-RNA interactions with an external force

Pierre Mangeol, Thierry Bizebard, Claude Chiaruttini, Marc Dreyfus, Mathias Springer, and Ulrich Bockelmann

Ribosomal (r-) RNA adopts a well-defined structure within the ribosome, but the role of r-proteins in stabilizing this structure is poorly understood. To address this issue, we use optical tweezers to unfold RNA fragments in the presence or absence of r-proteins. Here, we focus on Escherichia coli r-protein L20, whose globular C-terminal domain (L20C) recognizes an irregular stem in domain II of 23S rRNA. L20C also binds its own mRNA and represses its translation; binding occurs at two different sites - i.e., a pseudoknot and an irregular stem. We find that L20C makes rRNA and mRNA fragments encompassing its binding sites more resistant to mechanical unfolding. The regions of increased resistance correspond within two base pairs to the binding sites identified by conventional methods. While stabilizing specific RNA structures, L20C does not accelerate their formation from alternate conformations - i.e., it acts as a clamp but not as a chaperone. In the ribosome, L20C contacts only one side of its target stem but interacts with both strands, explaining its clamping effect. Other r-proteins bind rRNA similarly, suggesting that several rRNA structures are stabilized by "one-side" clamping.

DOI

Raman spectroscopy and microscopy based on mechanical force detection

I. Rajapaksa and H. Kumar Wickramasinghe

The Raman effect is typically observed by irradiating a sample with an intense light source and detecting the minute amount of frequency shifted scattered light. We demonstrate that Raman molecular vibrational resonances can be detected directly through an entirely different mechanism—namely, a force measurement. We create a force interaction through optical parametric down conversion between stimulated, Raman excited, molecules on a surface and a cantilevered nanometer scale probe tip brought very close to it. Spectroscopy and microscopy on clusters of molecules have been performed. Single molecules within such clusters are clearly resolved in the Raman micrographs. The technique can be readily extended to perform pump probe experiments for measuring inter- and intramolecular couplings and conformational changes at the single molecule level.

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Plasmonic Nanoparticle Chain in a Light Field: A Resonant Optical Sail

Silvia Albaladejo, Juan José Sáenz, and Manuel I. Marqués

Optical trapping and driving of small objects has become a topic of increasing interest in multidisciplinary sciences. We propose to use a chain made of metallic nanoparticles as a resonant light sail, attached by one end point to a transparent object and propelling it by the use of electromagnetic radiation. Driving forces exerted on the chain are theoretically studied as a function of radiation’s wavelength and chain’s alignments with respect to the direction of radiation. Interestingly, there is a window in the frequency spectrum in which null–torque equilibrium configuration, with minimum geometric cross section, corresponds to a maximum in the driving force.

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Optical trapping by a metal thin-film edge

Dongxiao Li, Yonggang Xi, and Hong Koo Kim

We present a new method of optical trapping based on the intensity gradient that is created by boundary diffraction of light at a metal thin-film edge. The structure consists of an optically thick metal-film step formed on a semi-transparent thin-film-metal-coated glass substrate. While the underlying thin layer of metal serves the purpose of suppressing the thermophoretic effect, the metal film step is found to induce a highly localized intensity distribution of light around the edge via self-interference of an incident wave and its boundary diffraction wave. Two-dimensional (2D) optical trapping of micron-sized dielectric particles is experimentally demonstrated with a 100-nm-thick Au film edge formed on a 10-nm-thick-Cr-coated glass slide. For a 2-µm polystyrene sphere, ∼2-pN trapping force is measured at 30-mW incident power of a 1064-nm laser beam. Not involving surface plasmon fields, this thin-film edge trapping is polarization independent and can be easily incorporated into an on-chip microfluidic configuration.

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Incandescent porous carbon microspheres to light up cells: solution phenomena and cellular uptake

Paul Duffy, Luís M. Magno, Rahul B. Yadav, Selene K. Roberts, Andrew D. Ward, Stanley W. Botchway, Paula E. Colavita and Susan J. Quinn

Carbon based materials are attractive for biological applications because of their excellent biocompatibility profile. Porous carbons with high specific surface area are particularly interesting because it is possible in principle to leverage their properties to deliver high drug payloads. In this work, porous carbon microspheres with high specific surface area were prepared and studied in solution and in cells. Raman optical tweezer trapping of microspheres, excited at 532 nm, results in graphitization and incandescence in solvents that display poor heat conduction. Fluorescence confocal microscopy imaging was used to demonstrate the uptake of fluorescently labelled microspheres by cells and the ability to leverage their optical absorptivity in order to cause carbon graphitization and cell death.

DOI

Optical trapping of porous silicon nanoparticles

Maria G Donato, Marco A Monaca, Giuliana Faggio, Luca De Stefano, Philip H Jones, Pietro G Gucciardi and Onofrio M Maragò

Silicon nanoparticles obtained by ball-milling of a 50% porosity silicon layer have been optically trapped when dispersed in a water–surfactant environment. We measured the optical force constants using linearly and radially polarized trapping beams finding a reshaping of the optical potential and an enhanced axial spring constant for the latter. These measurements open perspectives for the control and handling of silicon nanoparticles as labeling agents in biological analysis and fluorescence imaging techniques.

DOI

Nanoengineering a single-molecule mechanical switch using DNA self-assembly

Ken Halvorsen, Diane Schaak and Wesley P Wong

The ability to manipulate and observe single biological molecules has led to both fundamental scientific discoveries and new methods in nanoscale engineering. A common challenge in many single-molecule experiments is reliably linking molecules to surfaces, and identifying their interactions. We have met this challenge by nanoengineering a novel DNA-based linker that behaves as a force-activated switch, providing a molecular signature that can eliminate errant data arising from non-specific and multiple interactions. By integrating a receptor and ligand into a single piece of DNA using DNA self-assembly, a single tether can be positively identified by force–extension behavior, and receptor–ligand unbinding easily identified by a sudden increase in tether length. Additionally, under proper conditions the exact same pair of molecules can be repeatedly bound and unbound. Our approach is simple, versatile and modular, and can be easily implemented using standard commercial reagents and laboratory equipment. In addition to improving the reliability and accuracy of force measurements, this single-molecule mechanical switch paves the way for high-throughput serial measurements, single-molecule on-rate studies, and investigations of population heterogeneity.

DOI

Three-dimensional positioning of optically trapped nanoparticles

Takayuki Higuchi, Quang Duc Pham, Satoshi Hasegawa, and Yoshio Hayasaki

We firstly demonstrate the three-dimensional (3D) measurement of a nanometer-sized sphere held in optical tweezers in water using an in-line digital holographic microscope with a green light emitting diode. Suppressing the movement with optical tweezers enabled us to detect the three-dimensional position of a polystyrene sphere with a diameter of 200 nm. The positioning resolutions of the microscope were 3.2 nm in the transverse direction and 3.4 nm in the axial direction, from the standard deviation of measurements of the 200 nm sphere fixed on glass. Changes in the Brownian motion in response to a change in the trapping laser power were measured. We also demonstrated that this holographic measurement is an effective method for determining the threshold power of the optical trapping.

DOI

A novel video-based microsphere localization algorithm for low contrast silica particles under white light illumination

O. Ueberschär, C. Wagner, T. Stangner, C. Gutsche, F. Kremer

On the basis of a brief review of four common image recognition algorithms for microspheres made of polystyrene or melamine resin, we present a new microsphere localization method for low-contrast silica beads under white light illumination. We compare both the polystyrene and silica procedures with respect to accuracy and precision by means of an optical tweezers setup providing CMOS video microscopy capability. By that we demonstrate that our new silica algorithm achieves a relative position uncertainty of less than ±1 nm for micron-sized microspheres, significantly exceeding the precision of the other silica approaches studied. Second, we present an advancement of our single microsphere tracking method to scenarios where two polystyrene, melamine resin or silica microspheres are in close-to-contact proximity. While the majority of the analysis algorithms studied generate artefacts due to interference effects under these conditions, we show that our new approach yields accurate and precise results.

DOI

The Role of Tropomyosin Domains in Cooperative Activation of the Actin–Myosin Interaction

Yusuke Oguchi, Junji Ishizuka, Sarah E. Hitchcock-DeGregori, Shin'ichi Ishiwata, Masataka Kawai

To establish α-tropomyosin (Tm)'s structure–function relationships in cooperative regulation of muscle contraction, thin filaments were reconstituted with a variety of Tm mutants (Δ2Tm, Δ3Tm, Δ6Tm, P2sTm, P3sTm, P2P3sTm, P1P5Tm, and wtTm), and force and sliding velocity of the thin filament were studied using an in vitro motility assay. In the case of deletion mutants, Δ indicates which of the quasi-equivalent repeats in Tm was deleted. In the case of period (P) mutants, an Ala cluster was introduced into the indicated period to strengthen the Tm–actin interaction. In P1P5Tm, the N-terminal half of period 5 was substituted with that of period 1 to test the quasi-equivalence of these two Tm periods. The reconstitution included bovine cardiac troponin. Deletion studies revealed that period 3 is important for the positive cooperative effect of Tm on actin filament regulation and that period 2 also contributes to this effect at low ionic strength, but to a lesser degree. Furthermore, Tm with one extra Ala cluster at period 2 (P2s) or period 3 (P3s) did not increase force or velocity, whereas Tm with two extra Ala clusters (P2P3s) increased both force and velocity, demonstrating interaction between these periods. Most mutants did not move in the absence of Ca2+. Notable exceptions were Δ6Tm and P1P5Tm, which moved near at the full velocity, but with reduced force, which indicate impaired relaxation. These results are consistent with the mechanism that the Tm–actin interaction cooperatively affects actin to result in generation of greater force and velocity.

DOI

Controlled alignment of bacterial cells with oscillating optical tweezers

Gideon Carmon and Mario Feingold

We used optical tweezers to rotate bacterial cells relative to the optical axis. We rapidly oscillate the optical tweezers along an axis normal to the laser beam, thereby obtaining a linear trap. When the linear trap is longer than a trapped rod-shaped bacterial cell, the cell is aligned along the trap axis. Decreasing the length of the trap, we found that the cell rotates away from the image plane toward the optical axis. In the limit of a nonoscillating trap, the cell aligns along the optical axis. A defocused-edge detection method was devised to measure the orientation of the rotated cell from the corresponding phase-contrast images. Our technique can be used to image three-dimensional sub-cellular structures from different viewpoints and therefore may become a useful tool in fluorescence microscopy.

DOI

Applied Force Provides Insight into Transcriptional Pausing and Its Modulation by Transcription Factor NusA

Jing Zhou, Kook Sun Ha, Arthur La Porta, Robert Landick, Steven M. Block

Transcriptional pausing by RNA polymerase (RNAP) plays an essential role in gene regulation. Pausing is modified by various elongation factors, including prokaryotic NusA, but the mechanisms underlying pausing and NusA function remain unclear. Alternative models for pausing invoke blockade events that precede translocation (on-pathway), enzyme backtracking (off-pathway), or isomerization to a nonbacktracked, elemental pause state (off-pathway). We employed an optical trapping assay to probe the motions of individual RNAP molecules transcribing a DNA template carrying tandem repeats encoding the his pause, subjecting these enzymes to controlled forces. NusA significantly decreased the pause-free elongation rate of RNAP while increasing the probability of entry into short- and long-lifetime pauses, in a manner equivalent to exerting a ∼19 pN force opposing transcription. The effects of force and NusA on pause probabilities and lifetimes support a reaction scheme where nonbacktracked, elemental pauses branch off the elongation pathway from the pretranslocated state of RNAP.

DOI

Probing the mechanobiological properties of human embryonic stem cells in cardiac differentiation by optical tweezers

Youhua Tan, Chi-wing Kong, Shuxun Chen, Shuk Han Cheng, Ronald A. Li, Dong Sun

Human embryonic stem cells (hESC) and hESC-derived cardiomyocytes (hESC-CM) hold great promise for the treatment of cardiovascular diseases. However the mechanobiological properties of hESC and hESC-CM remains elusive. In this paper, we examined the dynamic and static micromechanical properties of hESC and hESC-CM, by manipulating via optical tweezers at the single-cell level. Theoretical approaches were developed to model the dynamic and static mechanical responses of cells during optical stretching. Our experiments showed that the mechanical stiffness of differentiated hESC-CM increased after cardiac differentiation. Such stiffening could associate with increasingly organized myofibrillar assembly that underlines the functional characteristics of hESC-CM. In summary, our findings lay the ground work for using hESC-CMs as models to study mechanical and contractile defects in heart diseases.

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Two-photon fluorescence diagnostics of femtosecond laser tweezers

Arijit Kumar De, Debjit Roy and Debabrata Goswami

We show how two-photon fluorescence signal can be used as an effective detection scheme for trapping particles of any size in comparison to methods using back-scattered light. Development of such a diagnostic scheme allows us a direct observation of trapping a single nanoparticle, which shows new directions to spectroscopy at the single-molecule level in solution.

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Measurement of viscosity of lyotropic liquid crystals by means of rotating laser-trapped microparticles

Qingkun Liu, Theodor Asavei, Taewoo Lee, Halina Rubinsztein-Dunlop, Sailing He, and Ivan I. Smalyukh

We describe a simple microrheology method to measure the viscosity coefficients of lyotropic liquid crystals. This approach is based on the use of a rotating laser-trapped optically anisotropic microsphere. In aligned liquid crystals that have negligible effect on trapping beam’s polarization, the optical torque is transferred from circularly polarized laser trapping beam to the optically anisotropic microparticle and creates the shear flow in the liquid crystalline fluid. The balance of optical and viscous torques yields the local effective viscosity coefficients of the studied lyotropic systems in cholesteric and lamellar phases. This simple yet powerful method is capable of probing viscosity of complex anisotropic fluids for small amounts of sample and even in the presence of defects that obstruct the use of conventional rheology techniques.

DOI

Robust control approach to force estimation in a constant position optical tweezers

Tanuj Aggarwal, Hullas Sehgal, and Murti Salapaka

Feedback enhanced optical tweezers with position regulation capability enable detection and estimation of forces in the pico-Newton regime. In this article we delineate the fundamental limitations and challenges of existing approaches for regulating position and force estimation in an optical tweezer. A modern control systems approach is shown to improve the bandwidth of force estimation by three to four times which is corroborated experimentally.

DOI

Multifocal optical trapping using counter-propagating radially-polarized beams

Yaoju Zhang, Yuxing Dai

A model of optical tweezers which can trap a chain of Rayleigh particles is proposed by using two counter-propagating equal highly focused radial polarized beams. Calculations show that a multifocal distribution along the optical axis is formed and the scattering force is equal to zero in the total focal filed, consequently a chain of metallic Rayleigh particles can be stably trapped. The trap force and the trap stiffness using two counter-propagating Radially-polarized beams are larger than those using two counter-propagating linearly-polarized beams. The trapping stability is calculated and analyzed in detail. The trapping number of particles in a trapping chain can be controlled by adjusting the aperture angle of the objective and the parameters of the filter used in the proposed trap system.

DOI

Inertial Effects of a Small Brownian Particle Cause a Colored Power Spectral Density of Thermal Noise

Anita Jannasch, Mohammed Mahamdeh, and Erik Schäffer

The random thermal force acting on Brownian particles is often approximated in Langevin models by a “white-noise” process. However, fluid entrainment results in a frequency dependence of this thermal force giving it a “color.” While theoretically well understood, direct experimental evidence for this colored nature of the noise term and how it is influenced by a nearby wall is lacking. Here, we directly measured the color of the thermal noise intensity by tracking a particle strongly confined in an ultrastable optical trap. All our measurements are in quantitative agreement with the theoretical predictions. Since Brownian motion is important for microscopic, in particular, biological systems, the colored nature of the noise and its distance dependence to nearby objects need to be accounted for and may even be utilized for advanced sensor applications.

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Chiral Self-Assembled Solid Microspheres: A Novel Multifunctional Microphotonic Device

Gabriella Cipparrone, Alfredo Mazzulla, Alfredo Pane, Raul Josue Hernandez, Roberto Bartolino

Solid chiral microspheres with unique and multifunctional optical properties are produced from cholesteric liquid crystal-water emulsions using photopolymerization processes. These self-organizing microspheres exhibit different internal configurations of helicoidal structures with radial, conical or cylindrical geometries, depending on the physicochemical characteristics of the precursor liquid crystal emulsion.

DOI

Mechanical stochastic tug-of-war models cannot explain bidirectional lipid-droplet transport

Ambarish Kunwar, Suvranta K. Tripathy, Jing Xu, Michelle K. Mattson, Preetha Anand, Roby Sigua, Michael Vershinin,Richard J. McKenney, Clare C. Yu, Alexander Mogilner, and Steven P. Gross

Intracellular transport via the microtubule motors kinesin and dynein plays an important role in maintaining cell structure and function. Often, multiple kinesin or dynein motors move the same cargo. Their collective function depends critically on the single motors’ detachment kinetics under load, which we experimentally measure here. This experimental constraint—combined with other experimentally determined parameters—is then incorporated into theoretical stochastic and mean-field models. Comparison of modeling results and in vitro data shows good agreement for the stochastic, but not mean-field, model. Many cargos in vivo move bidirectionally, frequently reversing course. Because both kinesin and dynein are present on the cargos, one popular hypothesis explaining the frequent reversals is that the opposite-polarity motors engage in unregulated stochastic tugs-of-war. Then, the cargos’ motion can be explained entirely by the outcome of these opposite-motor competitions. Here, we use fully calibrated stochastic and mean-field models to test the tug-of-war hypothesis. Neither model agrees well with our in vivo data, suggesting that, in addition to inevitable tugs-of-war between opposite motors, there is an additional level of regulation not included in the models.

DOI

Colored noise in the fluctuations of an extended DNA molecule detected by optical trapping

Ignacio A. Martínez, Saurabh Raj and Dmitri Petrov

We studied fluctuations of an optically trapped bead connected to a single DNA molecule anchored between the bead and a cover glass or between two optically trapped beads. Power spectral densities of the bead position for different extensions of the molecule were compared with the power spectral density of the position fluctuations of the same bead without the molecule attached. Experiments showed that the fluctuations of the DNA molecule extended up to 80% by a force of 3 pN include the colored noise contribution with spectral dependence 1/f α with α ∼ 0.75.

DOI

Momentum of light scattered from collections of particles

Zhisong Tong and Olga Korotkova

The angular dependence of the momentum flow of a polychromatic plane wave scattered from deterministic and random collections of particles is determined, within the occuracy of the first-order Born approximation, as a function of individual and collective properties of particles. The results are of importance for optimization of optical tweezers.

DOI

Optical Trapping of Beads and Jurkat Cells Using Micromachined Fresnel Zone Plate Integrated with Microfluidic Chip

Ju-Nan Kuo and Han-Zhong Hu

This paper presents a method for trapping beads and cells using a single-beam optical tweezer and a Fresnel zone plate integrated with a microfluidic chip. The experimental results show that a laser power of 2.4 mW is sufficient to trap 3-µm-diameter polystyrene beads, while a laser power of 1.5 mW is sufficient to trap individual Jurkat cells. The Fresnel zone plate developed in this study has many advantages, including a small size, a straightforward fabrication process, and a simple integration with microfluidic chips. Consequently, it provides an ideal solution for the trapping of a wide range of biological cells for analysis purposes.

DOI

Expanding the Optical Trapping Range of Lipid Vesicles to the Nanoscale

Poul M. Bendix and Lene B. Oddershede

Small unilamellar lipid vesicles with diameters down to 50 nm enclosing high refractive index sucrose cores can be optically trapped individually in three dimensions using a focused laser beam. Combined optical trapping and confocal microscopy allows for simultaneous quantitative measurements of the forces exerted on individual vesicles and of their size and shape. The position of individual vesicles in three dimensions is measured with nanometer spatial and 10 μs temporal resolution.

DOI

Holographic aberration correction: optimising the stiffness of an optical trap deep in the sample

Maria Dienerowitz, Graham Gibson, Richard Bowman, and Miles Padgett

We investigate the effects of 1st order spherical aberration and defocus upon the stiffness of an optical trap tens of μm into the sample. We control both these aberrations with a spatial light modulator. The key to maintain optimum trap stiffness over a range of depths is a specific non-trivial combination of defocus and axial objective position. This optimisation increases the trap stiffness by up to a factor of 3 and allows trapping of 1μm polystyrene beads up to 50μm deep in the sample.

DOI

Shear-flow-enhanced barrier crossing

Diego Kienle, Jochen Bammert, and Walter Zimmermann

We consider a single Brownian particle confined in a double well potential (DWP) and investigate its response to a linear shear flow by means of the probability density and current determined via numerical solution of the Fokker-Planck equation. Besides a shear-dependent distortion of the probability distribution, we find that the associated current crossing the potential barrier exhibits a convex dependency on the shear rate when the DWP's minima are far apart. With decreasing distance this functional dependency changes from a convex to concave characteristics accompanied with an increase of the probability current crossing the DWP's barrier. Through the difference map of the particle density distribution it is possible to extract the shear-flow-induced contribution to the particle density driving the barrier-crossing current. This may open the possibility to design specific flow profiles to optimize flow-induced activated transport of nanoparticles.

DOI

Optical manipulation of microparticles using whispering-gallery modes in a silicon nitride microdisk resonator

Hong Cai and Andrew W. Poon

We demonstrate optical manipulation of 1 μm sized polystyrene microparticles on silicon nitride microdisk resonator devices using whispering-gallery modes in an integrated optofluidic chip. We demonstrate multiple trapping tracks and extended trapping ranges within single wavelengths through exciting high-order modes. We observe various sets of trapping tracks and ranges through exciting various resonance modes. We switch particle traveling tracks by tuning the laser wavelength to various wavelengths. We also observe microparticles assembling along the trapping tracks.

DOI

The elementary events underlying force generation in neuronal lamellipodia

Ladan Amin, Erika Ercolini, Rajesh Shahapure, Giacomo Bisson & Vincent Torre

We have used optical tweezers to identify the elementary events underlying force generation in neuronal lamellipodia. When an optically trapped bead seals on the lamellipodium membrane, Brownian fluctuations decrease revealing the underlying elementary events. The distribution of bead velocities has long tails with frequent large positive and negative values associated to forward and backward jumps occurring in 0.1–0.2 ms with varying amplitudes up to 20 nm. Jump frequency and amplitude are reduced when actin turnover is slowed down by the addition of 25 nM Jasplakinolide. When myosin II is inhibited by the addition of 20 μM Blebbistatin, jump frequency is reduced but to a lesser extent than by Jasplainolide. These jumps constitute the elementary events underlying force generation.

DOI

Single Gradientless Light Beam Drags Particles as Tractor Beams

Andrey Novitsky, Cheng-Wei Qiu, and Haifeng Wang

Usually a light beam pushes a particle when the photons act upon it. We investigate the optical forces by nonparaxial gradientless beams and find that the forces can drag suitable particles all the way towards the light source. The major criterion of realizing the backward dragging force is the strong nonparaxiality of the light beam, which contributes to the pulling force owing to momentum conservation. The nonparaxiality of the Bessel beam can be manipulated to possess a dragging force along both the radial longitudinal directions, i.e., a “tractor beam” with stable trajectories is achieved.

DOI

Expanding the optical trapping range of lipid vesicles to the nano-scale

Poul Martin Bendix and Lene B Oddershede

Small unilamellar lipid vesicles with diameters down to 50 nm enclosing high refractive index sucrose cores can be optically trapped individually in three dimensions using a focused laser beam. Combined optical trapping and confocal microscopy allows for simultaneous quantitative measurements of the forces exerted on individual vesicles and of their size and shape. The position of individual vesicles in three dimensions is measured with nanometer spatial and ~10 μs temporal resolution.

DOI

Noise reduction by signal combination in Fourier space applied to drift correction in optical tweezers

Alireza Mashaghi, Peter J. Vach, and Sander J. Tans

A general method is proposed to reduce noise by combining signals. Different measurements of the same physical quantity often exhibit different noise levels in different frequency ranges. Hence, a single high-fidelity signal can be constructed by combining the low-noise parts of the signals in Fourier space. We demonstrate this method by reducing noise in the measured bead-to-bead distance in an optical tweezers setup.

DOI

Negative Nonconservative Forces: Optical “Tractor Beams” for Arbitrary Objects

S. Sukhov and A. Dogariu

Based on the conservation of linear momentum on scattering from arbitrary objects, we demonstrate the generation of nonconservative optical forces that act in a direction opposite to the propagation of the incident beam. The concept can be applied to tailor the force fields produced on nonabsorbing bodies regardless of their sizes and shapes.

DOI

Modeling of optical traps for aerosols

Daniel R. Burnham and David McGloin

Experimental observations suggest that there are differences between the behavior of particles optically trapped in air and trapped in a liquid phase. We have modified the Mie–Debye spherical aberration theory to numerically simulate an aerosol optical trap in an attempt to explain and predict the differences. The model incorporates Mie scattering and a trapping beam focused through media of stratified refractive index. We show that geometrical optics cannot correctly describe the aerosol optical trap and that spherical aberration must be included. We qualitatively explain the observed phenomena before discussing the limits of the experimental techniques and methods to improve it. We conclude that the system does not behave as a true “optical tweezers,” varying between levitation and single beam gradient force trapping, depending on particle and beam parameters.

DOI

Controlling the transverse momentum distribution of a light field via azimuth division of a hologram in holographic optical tweezers

Sheng-Yang Tseng and Long Hsu
This study proposes a method for creating a light field with controlled distribution of transverse momentum (TM) by displaying a hologram only in an azimuth region that centers at θ0 and has a range of Δθ of a spatial light modulator in holographic optical tweezers. This study utilized ray optics to analyze the TM of the resultant field, revealing that the direction of the TM is determined by the center angle of the azimuth region and that the magnitude of the TM is proportional to sin⁡(Δθ/2), without regarding the intensity. The relationship was verified experimentally. In addition, this study demonstrated moving particles along a designed path and depleting particles by the fields.

DOI

Single Nuclei Raman spectroscopy for Drug Evaluation

Hsin-Hung Lin , Yen-Chang Li , Chih-Hao Chang , Chun Liu , Alice L Yu , and Chung-Hsuan Chen

Detection of cellular changes at single-cell level has a great potential for biomedical and biopharmaceutical applications. Raman spectroscopy is an important tool for single-cell molecular imaging analysis. Raman spectroscopy can provide time-resolved information of the selected biomolecular distributions inside a single cell without the need of chemical labeling. In this study, we monitored the cellular responses to antineoplastic drug at a single cell basis with Raman spectroscopy. We demonstrated that single nuclei Raman spectroscopy has the ability to detect and identify nuclear changes related to cytotoxicity at lower concentrations and in shorter time span than conventional cell based assays. Thus, this strategy of using Raman spectroscopy of single, isolated nuclei may be very valuable for rapid and sensitive detection of cellular changes in response to chemotherapeutic agents.

DOI

Computational study of the optical trapping of ellipsoidal particles

Stephen H. Simpson and Simon Hanna

Ellipsoidal dielectric particles may be trapped in a linearly polarized Gaussian beam such that they are harmonically bound with respect to each of their rotational and translational degrees of freedom. The ellipsoid belongs to the highest symmetry class for which this is possible. Typically, the longest axis of the ellipsoid aligns itself with the incident beam axis and the second longest with the polarization direction. We investigate this special property by evaluating the trap stiffness matrix for dielectric ellipsoids with aspect ratios (largest:smallest dimension) in the range 1–10, using the discrete dipole approximation. The results are interpreted using a simple phenomenological model and conclusions are drawn concerning optimization of the trap stiffness for specific applications.

DOI

Optical tweezers setup with optical height detection and active height regulation under white light illumination

Carolin Wagner, Tim Stangner, Christof Gutsche, Olaf Ueberschär and Friedrich Kremer

An optical tweezers setup with optical detection in three dimensions and active height regulation has been developed. The presented novel method to determine the relative height of a microparticle from its microscopic image is based on the analysis of the integrated light intensity of the main maximum of the diffraction pattern. After the determination of a master curve as reference, the height can be detected with an accuracy of up to 2 nm. The method is applicable under microscopic white light illumination and is simple to implement. As an example of measurements where active height regulation is indispensable, force–distance curves are discussed. Furthermore, the colloid height is calculated geometrically. In the range where the geometrical estimation provides reliable results, the values are found to be in quantitative agreement with the suggested algorithm.

DOI

Attachment of Anti-GFP Antibodies to Microspheres for Optical Trapping Experiments

James A. Spudich, Sarah E. Rice, Ronald S. Rock, Thomas J. Purcell and Hans M. Warrick

In vitro motility assays enabled the analysis of coupling between ATP hydrolysis and movement of myosin along actin filaments or kinesin along microtubules. Single-molecule assays using laser trapping have been used to obtain more detailed information about kinesins, myosins, and processive DNA enzymes. The combination of in vitro motility assays with laser-trap measurements has revealed detailed dynamic structural changes associated with the ATPase cycle. This protocol describes a method for attaching anti-GFP (green fluorescent protein) antibodies to microspheres. GFP-motor fusion proteins can then be adsorbed to the microspheres for use in single-molecule motility studies and optical trapping experiments.

DOI

The Optical Trapping Dumbbell Assay for Nonprocessive Motors or Motors That Turn around Filaments

James A. Spudich, Sarah E. Rice, Ronald S. Rock, Thomas J. Purcell and Hans M. Warrick

In vitro motility assays enabled the analysis of coupling between ATP hydrolysis and movement of myosin along actin filaments or kinesin along microtubules. Single-molecule assays using laser trapping have been used to obtain more detailed information about kinesins, myosins, and processive DNA enzymes. The combination of in vitro motility assays with laser-trap measurements has revealed detailed dynamic structural changes associated with the ATPase cycle. This protocol describes the preparation of biotin–actin filaments and coverslips coated with polystyrene beads. These are then used in optical trapping dumbbell assays to study interactions between motors and filaments.

DOI

Optical Traps to Study Properties of Molecular Motors

James A. Spudich, Sarah E. Rice, Ronald S. Rock, Thomas J. Purcell and Hans M. Warrick

In vitro motility assays enabled the analysis of coupling between ATP hydrolysis and movement of myosin along actin filaments or kinesin along microtubules. Single-molecule assays using laser trapping have been used to obtain more detailed information about kinesins, myosins, and processive DNA enzymes. The combination of in vitro motility assays with laser-trap measurements has revealed detailed dynamic structural changes associated with the ATPase cycle. This article describes the use of optical traps to study processive and nonprocessive molecular motor proteins, focusing on the design of the instrument and the assays to characterize motility.

DOI

Detachment and reorientation of cells using near-infrared laser microbeam

Ling Gu, Samarendra K. Mohanty, Ninad Ingle

Reorientation of adhering cell(s) with respect to other cell(s) has not been yet possible, thus limiting study of controlled interaction between cells. Here, we report cell detachment upon irradiation with a focused near-infrared laser beam, and reorientation of adherent cells. The detached cell was transported along the axial direction by scattering force and trapped at a higher plane inside the media using the same laser beam by a gravito-optical trap. The trapped cell could then be repositioned by movement of the sample stage and reoriented by rotation of the astigmatic trapping beam. The height at which the cell was stably held was found to depend on the laser beam power. Viability of the detached and manipulated cell was found not to be compromised as confirmed by propidium iodide fluorescence exclusion assay. The reoriented cell was allowed to reattach to the substrate at a controlled distance and orientation with respect to other cells. Further, the cell was found to retain its shape even after multiple detachments and manipulation using the laser beam. This technique opens up new avenues for noncontact modification of cellular orientations that will enable study of intercellular interactions and design of engineered tissue.

DOI

CTGF/CCN2 has a chemoattractive function but a weak adhesive property to embryonic carcinoma cells

Diego P. Aguiar, Bruno Pontes, Fabio A. Mendes, Leonardo R. Andrade, Nathan B. Viana, José G. Abreu

Connective tissue growth factor (CTGF/CCN2) is a protein of the CCN family that modulates cell–ECM interactions in a variety of cell types. In this study, we investigated the chemotactic and adhesive properties of CCN2 protein in embryonic teratocarcinoma P19 cells. Initially, P19 cells were attracted to CCN2-coated agarose beads. In Boyden chamber experiments, CCN2-containing medium induced a threefold greater migration of P19 cells. CCN2 adhesion properties were studied by using optical tweezers. The specific adhesion times of P19 cells to polystyrene beads coated with laminin, fibronectin, CCN2and bovine serum albumin were 1.8 ± 0.5s, 2.7 ± 0.4s, 10 ± 2s and 13 ± 2s, respectively, revealing an unexpectedly low adhesive capacity of CCN2 protein for P19 cells. In conclusion, our findings support the chemoattractive role of CCN2 for P19 cells, but not its adhesive role when compared to laminin or fibronectin.

DOI

Probing ribosomal protein–RNA interactions with an external force

Pierre Mangeol, Thierry Bizebard, Claude Chiaruttini, Marc Dreyfus, Mathias Springer, and  Ulrich Bockelmann

Ribosomal (r-) RNA adopts a well-defined structure within the ribosome, but the role of r-proteins in stabilizing this structure is poorly understood. To address this issue, we use optical tweezers to unfold RNA fragments in the presence or absence of r-proteins. Here, we focus on Escherichia coli r-protein L20, whose globular C-terminal domain (L20C) recognizes an irregular stem in domain II of 23S rRNA. L20C also binds its own mRNA and represses its translation; binding occurs at two different sites—i.e., a pseudoknot and an irregular stem. We find that L20C makes rRNA and mRNA fragments encompassing its binding sites more resistant to mechanical unfolding. The regions of increased resistance correspond within two base pairs to the binding sites identified by conventional methods. While stabilizing specific RNA structures, L20C does not accelerate their formation from alternate conformations—i.e., it acts as a clamp but not as a chaperone. In the ribosome, L20C contacts only one side of its target stem but interacts with both strands, explaining its clamping effect. Other r-proteins bind rRNA similarly, suggesting that several rRNA structures are stabilized by “one-side” clamping.

DOI

The Complex Folding Network of Single Calmodulin Molecules

Johannes Stigler, Fabian Ziegler, Anja Gieseke, J. Christof M. Gebhardt, Matthias Rief

Direct observation of the detailed conformational fluctuations of a single protein molecule en route to its folded state has so far been realized only in silico. We have used single-molecule force spectroscopy to study the folding transitions of single calmodulin molecules. High-resolution optical tweezers assays in combination with hidden Markov analysis reveal a complex network of on- and off-pathway intermediates. Cooperative and anticooperative interactions across domain boundaries can be observed directly. The folding network involves four intermediates. Two off-pathway intermediates exhibit non-native interdomain interactions and compete with the ultrafast productive folding pathway.

DOI

Picoliter rheology of gaseous media using a rotating optically trapped birefringent microparticle

Yoshihiko Arita , Andrew W. McKinley , Michael Mazilu , Halina Rubinsztein-Dunlop , and Kishan Dholakia

An optically trapped birefringent microparticle is rotated by a circularly polarized beam in a confined gaseous medium. By recording the terminal rotation velocity and the change in polarization of the incident trapping beam, we determine the viscosity by probing a picolitervolume of air, carbon dioxide and argon in the vicinity of the microparticle. We also characterize the optical force acting on a trapped particle in air using the generalized Lorenz-Mie theory taking into account the aberrations present. This opens up a new potential application of optical tweezers for the accurate measurement of gas viscosity in confined geometries.

DOI

A microfluidic diffusion chamber for reversible environmental changes around flaccid lipid vesicles

Saša Vrhovec, Mojca Mally, Blaž Kavčič and Jure Derganc

The reversible environmental changes around flaccid lipid vesicles represent a considerable experimental challenge, particularly because of remarkable softness of flaccid membranes, which can warp irreversibly under the slightest hydrodynamic flow. As a result, we have developed a microfluidic device for the controlled analysis of individual flaccid, giant lipid vesicles in a changing chemical environment. The setup combines the advantages of a flow-free microfluidic diffusion chamber and optical tweezers, which are used to load the sample vesicles into the chamber. After a vesicle is loaded into the diffusion chamber, its chemical environment is controllably and reversibly changed solely by means of diffusion. The chamber is designed as a 250 micrometres-long and 100 micrometres-wide dead-end microchannel, which extends from a T-junction of the main microchannels. Measurements of the flow-velocity profile in the chamber show that the flow rate decreases exponentially and scales linearly with the flow rate in the main channel. The characteristic length of the exponential decrease is 15 (1 ± 0.13) micrometres, meaning that a large part of the diffusion chamber is effectively flow-free. The diffusion properties are assessed by monitoring the diffusion of a dye into the chamber. It was found that a simple 1D diffusion model fits well to the experimental data. The time needed for the exchange of solutes in the chamber is of the order of minutes, depending on the solute's molecular weight. Here, we demonstrate how the diffusion chamber can be used for reversible environmental changes around flaccid, giant lipid vesicles and membrane tethers (nanotubes).

DOI

Trapping, manipulation and rapid rotation of NBD-C8 fluorescent single microcrystals in optical tweezers

J. P. –Galaup, M. Rodríguez-Otazo, A. Augier-Calderín, J. –F. Lamère y S. Fery-Forges

We have built an optical tweezers experiment based on an inverted microscope to trap and manipulate single crystals of micro or sub-micrometer size made from fluorescent molecules of 4-octylamino-7-nitro-benzoxadiazole (NBD-C8). These single crystals have parallelepiped shapes and exhibit birefringence properties evidenced through optical experiments between crossed polarizers in a polarizing microscope. The crystals are uniaxial with their optical axis oriented along their largest dimension. Trapped in the optical trap, the organic micro-crystals are oriented in such a way that their long axis is along the direction o f the beam propagation, and their short axis follows the direction of the linear polarization. Therefore, with linearly polarized light, simply rotating the light polarization can orient the crystal. When using circularly or only elliptically polarized light, the crystal can spontaneously rotate and reach rotation speed of several hundreds of turns per second. A surprising result has been observed: when the incident power is growing up, the rotation speed increases to reach a maximum value and then decreases even when the power is still growing up. Moreover, this evolution is irreversible. Different possible explanations can be considered. The development of a 3D control of the crystals by dynamical holography using liquid crystal spatial modulators will be presented and discussed on the basis of the most recent results obtained.

DOI

Nonlinear Optical Processes in Optically Trapped InP Nanowires

Fan Wang, Peter J. Reece, Suriati Paiman, Qiang Gao, Hark Hoe Tan, and Chennupati Jagadish

We report on the observation of nonlinear optical excitation and related photoluminescence from single InP semiconductor nanowires held in suspension using a gradient force optical tweezers. Photoexcitation of free carriers is achieved through absorption of infrared (1.17 eV) photons from the trapping source via a combination of two- and three-photon processes. This was confirmed by power-dependent photoluminescence measurements. Marked differences in spectral features are noted between nonlinear optical excitation and direct excitation and are related to band-filling effects. Direct observation of second harmonic generation in trapped InP nanowires confirms the presence of nonlinear optical processes.

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Enhanced cell sorting and manipulation with combined optical tweezer and microfluidic chip technologies

Xiaolin Wang, Shuxun Chen, Marco Kong, Zuankai Wang, Kevin D. Costa, Ronald A. Li and Dong Sun

Sorting (or isolation) and manipulation of rare cells with high recovery rate and purity are of critical importance to a wide range of physiological applications. In the current paper, we report on a generic single cell manipulation tool that integrates optical tweezers and microfluidic chip technologies for handling small cell population sorting with high accuracy. The laminar flow nature of microfluidics enables the targeted cells to be focused on a desired area for cell isolation. To recognize the target cells, we develop an image processing methodology with a recognition capability of multiple features, e.g., cell size and fluorescence label. The target cells can be moved precisely by optical tweezers to the desired destination in a noninvasive manner. The unique advantages of this sorter are its high recovery rate and purity in small cell population sorting. The design is based on dynamic fluid and dynamic light pattern, in which single as well as multiple laser traps are employed for cell transportation, and a recognition capability of multiple cell features. Experiments of sorting yeast cells and human embryonic stem cells are performed to demonstrate the effectiveness of the proposed cell sorting approach.

DOI

Mechanical stability of low-humidity single DNA molecules

Silvia Hormeño, Borja Ibarra, José M. Valpuesta, José L. Carrascosa, J. Ricardo Arias-Gonzalez

DNA electrostatic character is mostly determined by both water and counterions activities in the phosphate backbone which, together with base sequence, further confer its higher-order structure. We overstretch individual double-stranded DNA molecules in water-ethanol solutions to investigate the modulation of its mechanical stability by hydration and polycations. We find that DNA denatures as ethanol concentration is increased and spermine concentration decreased. This is manifested by an increase in melting hysteresis between the stretch and release curves, with sharp transition at 10% ethanol and reentrant behavior at 60%, by a loss of cooperativity in the overstretching transition and by a dramatic decrease of both the persistence length and the flexural rigidity. Changes in base-stacking stability which are characteristic of the B-A transition between 70% and 80% ethanol concentration do not manifest in the mechanical properties of the double-helical molecule at low or high force or in the behavior of the overstretching and melting transitions within this ethanol concentration range. This is consistent with a mechanism in which A-type base-stacking is unstable in the presence of tension. Binding of motor proteins to DNA locally reduces the number of water molecules and therefore, our results may shed light on analogous reduced-water activity of DNA conditions caused by other molecules which interact with DNA in vivo.

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Plasmon-Exciton Interactions on Single Thermoresponsive Platforms Demonstrated by Optical Tweezers

Silvia Hormeño, Neus G. Bastús, Andrea Pietsch, Horst Weller, J. R. Arias-Gonzalez, and Beatriz H. Juárez

Optical and hydrodynamic-size studies on single bare thermo-responsive microspheres, and microspheres covered either with Au nanoparticles, CdSe/CdS quantum dots, or a combination of both have been performed by optical tweezers. The photothermal heating of water in the focal region boosts the shrinkage of the microspheres, an effect that is intensified in the presence of Au nanoparticles. In contrast, bigger microspheres are measured when they are covered with quantum dots. Plasmon-exciton interactions are observable in the trap in the combined Au and quantum dots hybrid systems.

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Planar silicon microrings as wavelength-multiplexed optical traps for storing and sensing particles

Shiyun Lin and Kenneth B. Crozier

We demonstrate the trapping of particles with silicon microring resonators integrated with waveguides. Multiple microrings with different resonant wavelengths are integrated with each waveguide. We demonstrate that tuning the laser wavelength to the resonance wavelengths of different rings enables trapped particles to be transferred back and forth between the rings. We demonstrate that the change in output power arising from particle-induced resonance shift enables the real-time monitoring of trapped particles, such as their number and velocities, without the need for an external imaging system. The techniques we describe here could form the basis for small footprint systems in which objects are moved between multiple locations on a chip, at each of which different operations are performed and the objects' properties sensed.

DOI